Methods for manufacturing t cells

ABSTRACT

The disclosure relates to methods of manufacturing T cells for adoptive immunotherapy. The disclosure further provides for methods of genetically transducing T cells, methods of using T cells, and T cell populations thereof. In an aspect, the disclosure provides for methods of thawing frozen peripheral blood mononuclear cells (PBMC), resting the thawed PBMC, activating the T cell in the cultured PBMC with an anti-CD3 antibody and an anti-CD28 antibody immobilized on a solid phase, transducing the activated T cell with a viral vector, expanding the transduced T cell, and obtaining expanded T cells.

CROSS REFERENCE TO RELATED APPLICATIONS

This application is a continuation of U.S. patent application Ser. No.16/271,393, filed 8 Feb. 2019, which claims priority to U.S. Provisionalapplication No. 62/726,350, filed on Sep. 3, 2018, U.S. provisionalapplication No. 62/647,571, filed on Mar. 23, 2018, U.S. provisionalapplication No. 62/633,113, filed on Feb. 21, 2018, U.S. provisionalapplication No. 62/628,521, filed on Feb. 9, 2018, German PatentApplication number 10 2018 108 996.1, filed Apr. 16, 2018; German PatentApplication number 10 2018 104 628.6, filed Feb. 28, 2018; and GermanPatent Application number 10 2018 102 971.3, filed Feb. 9, 2018, thecontents of each are hereby incorporated by reference in theirentireties.

REFERENCE TO SEQUENCE LISTING SUBMITTED AS A COMPLIANT ASCII TEXT FILE(.TXT)

Pursuant to the EFS-Web legal framework and 37 CFR §§ 1.821-825 (seeMPEP § 2442.03(a)), a Sequence Listing in the form of an ASCII-complianttext file (entitled “Sequence_Listing_3000011-006005_ST25.txt” createdon 1 Oct. 2020, and 24,767 bytes in size) is submitted concurrently withthe instant application, and the entire contents of the Sequence Listingare incorporated herein by reference.

FIELD

The present disclosure generally relates to methods of manufacturing Tcells for adoptive immunotherapy. The disclosure further provides formethods of genetically transducing T cells, methods of using T cells,and T cell populations thereof.

BACKGROUND

Redirecting the specificity of T cells against tumor-associated antigensby genetically enforced expression of T cell receptors (TCRs) orchimeric antigen receptor (CARs) has recently boosted the field ofadoptive T cell transfer. The use of second- and third-generation CARshas helped to resolve the long-standing problem of insufficient in vivoT cell persistence after transfer that was severely hampering itsefficacy. Nevertheless, important obstacles for a wider applicationremain, such as the necessity to produce T cell products on anindividualized basis, making this promising treatment approach hardlyeconomically feasible. Although the use of T cells, for exampleautologous T cells, has shown promise, it can be difficult to obtain asuitable numbers of autologous cells in heavily pretreated patients.

U.S. 2003/0170238 and U.S. 2003/0175272 describe methods for adoptiveimmunotherapy, in which T cells are allowed to rest by removing themfrom activation stimuli for at least 48-72 hours, typically at leastabout 72-120 hours, and then reactivating them prior to infusion bylabeling cells, for example, with mitogenic monoclonal antibodies(mAbs), such as soluble anti-CD3 and anti-CD28 mAbs, and then mixing thelabeled cells with autologous mononuclear cells that are optionallyenhanced in monocytes and granulocytes.

U.S. 2017/0051252 describes methods for manufacturing T celltherapeutics including the steps of obtaining a population of cellscontaining T cells and antigen presenting cells (APCs); culturing thepopulation of cells in a cell culture medium comprising (i) one or morecytokines, (ii) an anti-CD3 antibody or CD3-binding fragment thereof,and (iii) an anti-CD28 antibody or a CD28-binding fragment thereof, B7-1or a CD28-binding fragment thereof, or B7-2 or a CD28-binding fragmentthereof, in which the culture activates and stimulates the T cells;transducing the population of activated cells with a viral vector; andculturing the population of cells in a cell growth medium to expand thetransduced T cells; thereby manufacturing T cell therapeutics.

Improved strategies are needed for transducing cell populations in vitrothat could generate enough T cells for research, diagnostic, andtherapeutic purposes. A solution to this technical problem is providedherein.

BRIEF SUMMARY

In an aspect, the present disclosure relates to a method of transducinga T cell including thawing frozen peripheral blood mononuclear cells(PBMC), resting the thawed PBMC, activating the T cell in the culturedPBMC with an anti-CD3 antibody and an anti-CD28 antibody, transducingthe activated T cell with a viral vector, expanding the transduced Tcell, and obtaining the expanded T cells.

In an aspect, the T cell is activated in cultured PBMC with an anti-CD3antibody and an anti-CD28 antibody immobilized on a solid phase support.

In another aspect, the resting step may be carried out within a periodof no more than about 1 hour, no more than about 2 hours, no more thanabout 3 hours, no more than about 4 hours, no more than about 5 hours,no more than about 6 hours, no more than about 7 hours, no more thanabout 8 hours, no more than about 9 hours, no more than about 10 hours,no more than about 11 hours, no more than about 12 hours, no more thanabout 18 hours, no more than about 24 hours, no more than about 48hours, no more than about 36 hours, no more than about 48 hours, no morethan about 60 hours, no more than about 72 hours, no more than about 84hours, no more than about 96 hours, no more than about 108 hours, or nomore than about 120 hours.

In another aspect, resting may be carried out within a period of fromabout 0.5 hour to about 48 hours, about 0.5 hour to about 36 hours,about 0.5 hour to about 24 hours, about 0.5 hour to about 18 hours,about 0.5 hour to about 12 hours, about 0.5 hour to about 6 hours, about1 hour to about 6 hours, about 2 hours to about 5 hours, about 3 hoursto about 5 hours, about 3 hours to about 4 hours, about 4 to about 5hours, or about 1 hours to about 24 hours, about 2 to about 24 hours,about 12 to about 48 hours, about 0.5 hour to about 120 hours, about 0.5hour to about 108 hours, about 0.5 hour to about 96 hours, about 0.5hour to about 84 hours, about 0.5 hour to about 72 hours, or about 0.5hour to about 60 hours.

In another aspect, the resting step may be carried out within a periodof about 1 hour, about 2 hours, about 3 hours, about 4 hours, about 5hours, about 6 hours, about 7 hours, about 8 hours, about 9 hours, orabout 10 hours.

In an aspect, the fold expansion of T cells produced with a resting stepof about 1 hour, about 2 hours, about 3 hours, about 4 hours, about 5hours, about 6 hours, about 7 hours, about 8 hours, about 9 hours, about10 hours, about 2 hours to about 5 hours, about 3 hours to about 5hours, about 3 hours to about 4 hours, or about 4 to about 5 hours isabout equal to (about 1:1); about at least 1.1 times, about at least 1.2times, about at least 1.3 times, about at least 1.5 times, about atleast 1.7 times, or about at least 2.0 times greater than the foldexpansion of T cells produced with a resting step of about 16 hours,about 17 hours, about 18 hours, about 19 hours, about 20 hours, about 24hours, or about 16 to about 20 hours. In a preferred aspect, the foldexpansion of T cells produced with a resting step of about 4 hours isabout at least 1.5 times greater than the fold expansion of T cellsproduced with a resting step of about 16 hours (for example, overnight).In an aspect, the only difference between the production of the T cellsis the reduced resting time.

In an aspect, the number of T cells produced with a resting step ofabout 1 hour, about 2 hours, about 3 hours, about 4 hours, about 5hours, about 6 hours, about 7 hours, about 8 hours, about 9 hours, about10 hours, about 2 hours to about 5 hours, about 3 hours to about 5hours, about 3 hours to about 4 hours, or about 4 to about 5 hours isabout equal to (about 1:1); about at least 1.1 times, about at least 1.2times, about at least 1.3 times, about at least 1.5 times, about atleast 1.7 times, or about at least 2.0 times greater than the number ofT cells produced with a resting step of about 16 hours, about 17 hours,about 18 hours, about 19 hours, about 20 hours, about 24 hours, or about16 to about 20 hours. In a preferred aspect, the number of T cellsproduced with a resting step of about 4 hours is about at least 1.5times or about 1.3 times to about 2.0 times greater than the foldexpansion of T cells produced with a resting step of about 16 hours (forexample, overnight). In an aspect, the only difference between theproduction of the T cells is the reduced resting time.

In yet another aspect, anti-CD3 antibody and the anti-CD28 antibody eachmay have a concentration of no more than about 0.1 μg/ml, no more thanabout 0.2 μg/ml, no more than about 0.3 μg/ml, no more than about 0.4μg/ml, no more than about 0.5 μg/ml, no more than about 0.6 μg/ml, nomore than about 0.7 μg/ml, no more than about 0.8 μg/ml, no more thanabout 0.9 μg/ml, no more than about 1.0 μg/ml, no more than about 2.0μg/ml, no more than about 4.0 μg/ml, no more than about 6.0 μg/ml, nomore than about 8.0 μg/ml, or no more than about 10.0 μg/ml.

In yet another aspect, anti-CD3 antibody and the anti-CD28 antibody eachmay have a concentration of from about 0.1 μg/ml to about 1.0 μg/ml,about 0.1 μg/ml to about 0.8 μg/ml, about 0.1 μg/ml to about 0.6 μg/ml,about 0.1 μg/ml to about 0.5 μg/ml, about 0.1 μg/ml to about 0.25 μg/ml,about 0.2 μg/ml to about 0.5 μg/ml, about 0.2 μg/ml to about 0.3 μg/ml,about 0.3 μg/ml to about 0.5 μg/ml, about 0.3 μg/ml to about 0.4 μg/ml,about 0.2 μg/ml to about 0.5 μg/ml, about 0.1 μg/ml to about 10.0 μg/ml,about 0.1 μg/ml to about 8.0 μg/ml, about 0.1 μg/ml to about 6.0 μg/ml,about 0.1 μg/ml to about 4.0 μg/ml, or about 0.1 μg/ml to about 2.0μg/ml.

In an aspect, activation described herein may be carried out within aperiod of no more than about 1 hour, no more than about 2, hours, nomore than about 3 hours, no more than about 4 hours, no more than about5 hours, no more than about 6 hours, no more than about 7 hours, no morethan about 8 hours, no more than about 9 hours, no more than about 10hours, no more than about 11 hours, no more than about 12 hours, no morethan about 14 hours, no more than about 16 hours, no more than about 18hours, no more than about 20 hours, no more than about 22 hours, no morethan about 24 hours, no more than about 26 hours, no more than about 28hours, no more than about 30 hours, no more than about 36 hours, no morethan about 48 hours, no more than about 60 hours, no more than about 72hours, no more than about 84 hours, no more than about 96 hours, no morethan about 108 hours, or no more than about 120 hours.

In another aspect, activation described herein may be carried out withina period of from about 1 hour to about 120 hours, about 1 hour to about108 hours, about 1 hour to about 96 hours, about 1 hour to about 84hours, about 1 hour to about 72 hours, about 1 hour to about 60 hours,about 1 hour to about 48 hours, about 1 hour to about 36 hours, about 1hour to about 24 hours, about 2 hours to about 24 hours, about 4 hoursto about 24 hours, about 6 hours to about 24 hours, about 8 hours toabout 24 hours, about 10 hours to about 24 hours, about 12 hours toabout 24 hours, about 12 hours to about 72 hours, about 24 hours toabout 72 hours, about 6 hours to about 48 hours, about 24 hours to about48 hours, about 6 hours to about 72 hours, or about 1 hours to about 12hours.

In an aspect, T cells described herein are autologous to the patient orindividual. In another aspect, T cells described herein are allogenic tothe patient or individual.

In another aspect, a solid phase described herein may be a surface of abead, a plate, a flask, or a bag.

In yet another aspect, a plate described herein may be a 6-well,12-well, or 24-well plate.

In an aspect, a flask described herein may have a seeding surface areaof at least about 25 cm², about 75 cm², about 92.6 cm², about 100 cm²,about 150 cm², about 162 cm², about 175 cm², about 225 cm², about 235cm², about 300 cm², about 1720 cm², about 25 cm² to about 75 cm², about25 cm² to about 225 cm², or about 25 cm² to about 1720 cm².

In another aspect, a bag described herein may have a volume of fromabout 50 ml to about 100 liters, about 100 ml to about 100 liters, about150 ml to about 100 liters, about 200 ml to about 100 liters, about 250ml to about 100 liters, about 500 ml to about 100 liters, about 1 literto about 100 liters, about 1 liter to about 75 liters, about 1 liter toabout 50 liters, about 1 liter to about 25 liters, about 1 liter toabout 20 liters, about 1 liter to about 15 liters, about 1 liter toabout 10 liters, about 1 liter to about 5 liters, about 1 liter to about2.5 liters, or about 1 liter to about 2 liters.

In yet another aspect, activation described herein may be carried out inthe presence of the T cell activation stimulus.

In an aspect, cytokines described herein may include interleukin 2(IL-2), interleukin 7 (IL-7), interleukin 15 (IL-15), and/or interleukin21 (IL-21).

In another aspect, the concentration of IL-7 may be no more than about 1ng/ml, no more than about 2 ng/ml, no more than about 3 ng/ml, no morethan about 4 ng/ml, no more than about 5 ng/ml, no more than about 6ng/ml, no more than about 7 ng/ml, no more than about 8 ng/ml, no morethan about 9 ng/ml, no more than about 10 ng/ml, no more than about 11ng/ml, no more than about 12 ng/ml, no more than about 13 ng/ml, no morethan about 14 ng/ml, no more than about 15 ng/ml, no more than about 16ng/ml, no more than about 17 ng/ml, no more than about 18 ng/ml, no morethan about 19 ng/ml, no more than about 20 ng/ml, no more than about 25ng/ml, no more than about 30 ng/ml, no more than about 35 ng/ml, no morethan about 40 ng/ml, no more than about 45 ng/ml, no more than about 50ng/ml, no more than about 60 ng/ml, no more than about 70 ng/ml, no morethan about 80 ng/ml, no more than about 90 ng/ml, or no more than about100 ng/ml.

In another aspect, the concentration of IL-7 may be from about 1 ng/mlto 100 ng/ml, about 1 ng/ml to 90 ng/ml, about 1 ng/ml to 80 ng/ml,about 1 ng/ml to 70 ng/ml, about 1 ng/ml to 60 ng/ml, about 1 ng/ml to50 ng/ml, about 1 ng/ml to 40 ng/ml, about 1 ng/ml to 30 ng/ml, about 1ng/ml to 20 ng/ml, about 1 ng/ml to 15 ng/ml, about 1 ng/ml to 10 ng/ml,about 2 ng/ml to 10 ng/ml, about 4 ng/ml to 10 ng/ml, about 6 ng/ml to10 ng/ml, or about 5 ng/ml to 10 ng/ml.

In yet another aspect, the concentration of IL-15 may be no more thanabout 5 ng/ml, no more than about 10 ng/ml, no more than about 15 ng/ml,no more than about 20 ng/ml, no more than about 25 ng/ml, no more thanabout 30 ng/ml, no more than about 35 ng/ml, no more than about 40ng/ml, no more than about 45 ng/ml, no more than about 50 ng/ml, no morethan about 60 ng/ml, no more than about 70 ng/ml, no more than about 80ng/ml, no more than about 90 ng/ml, no more than about 100 ng/ml, nomore than about 110 ng/ml, no more than about 120 ng/ml, no more thanabout 130 ng/ml, no more than about 140 ng/ml, no more than about 150ng/ml, 200 ng/ml, 250 ng/ml, 300 ng/ml, 350 ng/ml, 400 ng/ml, 450 ng/ml,or 500 ng/ml.

In another aspect, the concentration of IL-15 may be from about 5 ng/mlto 500 ng/ml, about 5 ng/ml to 400 ng/ml, about 5 ng/ml to 300 ng/ml,about 5 ng/ml to 200 ng/ml, about 5 ng/ml to 150 ng/ml, about 5 ng/ml to100 ng/ml, about 10 ng/ml to 100 ng/ml, about 20 ng/ml to 100 ng/ml,about 30 ng/ml to 100 ng/ml, about 40 ng/ml to 100 ng/ml, about 50 ng/mlto 100 ng/ml, about 60 ng/ml to 100 ng/ml, about 70 ng/ml to 100 ng/ml,about 80 ng/ml to 100 ng/ml, about 90 ng/ml to 100 ng/ml, about 1 ng/mlto 50 ng/ml, about 5 ng/ml to 50 ng/ml, about 10 ng/ml to 50 ng/ml, orabout 20 ng/ml to 50 ng/ml.

In another aspect, the concentration of IL-2 may be no more than about1000 IU/ml, no more than about 950 IU/ml, no more than about 900 IU/ml,no more than about 850 IU/ml, no more than about 800 IU/ml, no more thanabout 750 IU/ml, no more than about 700 IU/ml, no more than about 650IU/ml, no more than about 600 IU/ml, no more than about 550 IU/ml, nomore than about 500 IU/ml, no more than about 450 IU/ml, no more thanabout 400 IU/ml, no more than about 350 IU/ml, no more than about 300IU/ml, no more than about 250 IU/ml, no more than about 200 IU/ml, nomore than about 150 IU/ml, no more than about 100 IU/ml, no more thanabout 90 IU/ml, no more than about 80 IU/ml, no more than about 70IU/ml, no more than about 65 IU/ml, no more than about 60 IU/ml, no morethan about 55 IU/ml, no more than about 50 IU/ml, no more than about 40IU/ml, no more than about 30 IU/ml, no more than about 20 IU/ml, no morethan about 10 IU/ml, or no more than about 5 IU/ml.

In another aspect, the concentration of IL-2 may be from about 10 IU/mlto 1000 IU/ml, about 20 IU/ml to 900 IU/ml, about 30 IU/ml to 800 IU/ml,about 40 IU/ml to 700 IU/ml, about 50 IU/ml to 600 IU/ml, about 50 IU/mlto 550 IU/ml, about 50 IU/ml to 500 IU/ml, about 50 IU/ml to 450 IU/ml,about 50 IU/ml to 400 IU/ml, about 50 IU/ml to 350 IU/ml, about 50 IU/mlto 300 IU/ml, about 50 IU/ml to 250 IU/ml, about 50 IU/ml to 200 IU/ml,about 50 IU/ml to 150 IU/ml, or about 50 IU/ml to 100 IU/ml.

In another aspect, the concentration of IL-21 may be no more than about1 ng/ml, no more than about 2 ng/ml, no more than about 3 ng/ml, no morethan about 4 ng/ml, no more than about 5 ng/ml, no more than about 6ng/ml, no more than about 7 ng/ml, no more than about 8 ng/ml, no morethan about 9 ng/ml, no more than about 10 ng/ml, no more than about 11ng/ml, no more than about 12 ng/ml, no more than about 13 ng/ml, no morethan about 14 ng/ml, no more than about 15 ng/ml, no more than about 16ng/ml, no more than about 17 ng/ml, no more than about 18 ng/ml, no morethan about 19 ng/ml, no more than about 20 ng/ml, no more than about 25ng/ml, no more than about 30 ng/ml, no more than about 35 ng/ml, no morethan about 40 ng/ml, no more than about 45 ng/ml, no more than about 50ng/ml, no more than about 60 ng/ml, no more than about 70 ng/ml, no morethan about 80 ng/ml, no more than about 90 ng/ml, or no more than about100 ng/ml.

In another aspect, the concentration of IL-21 may be from about 1 ng/mlto 100 ng/ml, about 1 ng/ml to 90 ng/ml, about 1 ng/ml to 80 ng/ml,about 1 ng/ml to 70 ng/ml, about 1 ng/ml to 60 ng/ml, about 1 ng/ml to50 ng/ml, about 1 ng/ml to 40 ng/ml, about 1 ng/ml to 30 ng/ml, about 1ng/ml to 20 ng/ml, about 1 ng/ml to 15 ng/ml, about 1 ng/ml to 10 ng/ml,about 2 ng/ml to 10 ng/ml, about 4 ng/ml to 10 ng/ml, about 6 ng/ml to10 ng/ml, about 5 ng/ml to 10 ng/ml, about 10 ng/ml to 20 ng/ml, about10 ng/ml to 30 ng/ml, about 10 ng/ml to 40 ng/ml, about 10 ng/ml to 50ng/ml, about 10 ng/ml to 60 ng/ml, about 10 ng/ml to 70 ng/ml, about 10ng/ml to 80 ng/ml, about 10 ng/ml to 90 ng/ml, or about 10 ng/ml to 100ng/ml.

In an aspect, transducing described herein may be carried out within aperiod of no more than about 1 hour, no more than about 2 hours, no morethan about 3 hours, no more than about 4 hours, no more than about 5hours, no more than about 6 hours, no more than about 7 hours, no morethan about 8 hours, no more than about 9 hours, no more than about 10hours, no more than about 11 hours, no more than about 12 hours, no morethan about 14 hours, no more than about 16 hours, no more than about 18hours, no more than about 20 hours, no more than about 22 hours, no morethan about 24 hours, no more than about 26 hours, no more than about 28hours, no more than about 30 hours, no more than about 36 hours, no morethan about 42 hours, no more than about 48 hours, no more than about 54hours, no more than about 60 hours, no more than about 66 hours, no morethan about 72 hours, no more than about 84 hours, no more than about 96hours, no more than about 108 hours, or no more than about 120 hours.

In yet another aspect, transducing described herein may be carried outwithin a period of from about 1 hour to about 120 hours, about 1 hour toabout 108 hours, about 1 hour to about 96 hours, about 1 hour to about72 hours, about 1 hour to about 48 hours, about 1 hour to about 36hours, about 1 hour to about 24 hours, about 1 hour to about 12 hours,about 2 hours to about 24 hours, about 4 hours to about 24 hours, about12 hours to about 24 hours, about 12 hours to about 48 hours, about 12hour to about 72 hours, about 24 hours to about 72 hours, or about 36hours to about 72 hours.

In another aspect, viral vector described herein may be a γ-retroviralvector expressing a T cell receptor (TCR).

In yet another aspect, viral vector described herein may be a lentiviralvector expressing a TCR.

In an aspect, transducing described herein may be carried out in thepresence of the T cell activation stimulus.

In an aspect, expanding described herein may be carried out in thepresence of the T cell activation stimulus.

In an aspect, expanding described herein may be carried out within aperiod of no more than about 1 day, no more than about 2 days, no morethan about 3 days, no more than about 4 days, no more than about 5 days,no more than about 6 days, no more than about 7 days, no more than about8 days, no more than about 9 days, no more than about 10 days, no morethan about 15 days, no more than about 20 days, no more than about 25days, or no more than about 30 days.

In another aspect, expanding described herein may be carried out withina period of from about 1 day to about 30 days, about 1 day to about 25days, about 1 day to about 20 days, about 1 day to about 15 days, about1 day to about 10 days, about 2 days to about 10 days, about 3 days toabout 10 days, about 4 days to about 10 days, about 4 days to about 30days, about 6 days to about 25 days, about 10 days to about 30 days, orabout 12 days to about 30 days.

In an aspect, the number of the obtained T cells may be at least about1×10⁹, may be at least about 2×10⁹, may be at least about 3×10⁹, may beat least about 4×10⁹, may be at least about 5×10⁹, may be at least about6×10⁹, may be at least about 7×10⁹, may be at least about 8×10⁹, may beat least about 9×10⁹, may be at least about 1×10¹⁰, may be at leastabout 5×10¹⁰, may be at least about 1×10¹¹, may be at least about5×10¹¹, may be at least about 1×10¹², may be at least about 5×10¹² ormay be at least about 1×10¹³ cells.

In another aspect, the number of the obtained T cells may be from about1×10⁹ to about 1×10¹³, about 1×10⁹ to about 5×10¹², about 1×10⁹ to about1×10¹², about 1×10⁹ to about 5×10¹¹, about 1×10⁹ to about 1×10¹¹, about1×10⁹ to about 5×10¹⁰, about 1×10⁹ to about 1×10¹⁰, about 2×10⁹ to about1×10¹⁰, about 3×10⁹ to about 1×10¹⁰, about 4×10⁹ to about 1×10¹⁰, about5×10⁹ to about 1×10¹⁰, about 6×10⁹ to about 1×10¹⁰, about 7×10⁹ to about1×10¹⁰, about 8×10⁹ to about 1×10¹⁰, or about 9×10⁹ to about 1×10¹⁰cells.

In an aspect, the obtained T cells may be a CD3⁺ CD8⁺ T cell and/or CD3⁺CD4+ T cells.

In another aspect, PBMC may be obtained from the patient.

In yet another aspect, the present disclosure relates to geneticallytransduced T cells produced by the method described herein.

In another aspect, the present disclosure relates to pharmaceuticalcompositions containing the genetically transduced T cells produced bythe method described herein and pharmaceutically acceptable carriers.

In another aspect, the present disclosure relates to a method ofpreparing a T cell population, including thawing frozen peripheral bloodmononuclear cells (PBMC), resting the thawed PBMC, activating the T cellin the rested PBMC with an anti-CD3 antibody and an anti-CD28 antibodyimmobilized on a solid phase, expanding the activated T cell, andobtaining the T cell population comprising the expanded T cell.

In yet another aspect, the present disclosure relates to a T cellpopulation prepared by the method described herein.

In another aspect, the present disclosure relates to methods of treatinga patient or individual having a cancer or in need of a treatmentthereof, comprising administering to the patient an effective amount ofthe expanded T cells described herein. In an aspect, the patient orindividual in need thereof is a cancer patient. In an aspect, the cancerto be treated is selected from one or more of hepatocellular carcinoma(HCC), colorectal carcinoma (CRC), glioblastoma (GB), gastric cancer(GC), esophageal cancer, non-small cell lung cancer (NSCLC), pancreaticcancer (PC), renal cell carcinoma (RCC), benign prostate hyperplasia(BPH), prostate cancer (PCA), ovarian cancer (OC), melanoma, breastcancer, chronic lymphocytic leukemia (CLL), Merkel cell carcinoma (MCC),small cell lung cancer (SCLC), Non-Hodgkin lymphoma (NHL), acute myeloidleukemia (AML), gallbladder cancer and cholangiocarcinoma (GBC, CCC),urinary bladder cancer (UBC), acute lymphocytic leukemia (ALL), anduterine cancer (UEC).

In another aspect, the expanding may be carried out in the presence ofat least one cytokine selected from the group consisting of IL-2, IL-7,IL-12, IL-15, and IL-21. In an aspect, the expansion takes place in thepresence of a combination IL-7 and IL-15.

In another aspect, the thawing, the resting, the activating, thetransducing, the expanding, and/or the obtaining may be performed in aclosed system.

In another aspect, the present disclosure relates to a method ofpreparing a T cell population, including obtaining fresh peripheralblood mononuclear cells (PBMC), i.e., PBMC is not obtained by thawingcryopreserved PBMC, activating the T cell in the fresh PBMC with ananti-CD3 antibody and an anti-CD28 antibody, transducing the activated Tcell with a viral vector, expanding the transduced T cell, andharvesting the expanded T cell.

In an aspect, the obtaining and the activating may be performed for nomore than 1 day.

In an aspect, the expanding may be performed for more than 1 day.

In another aspect, the expanding may be performed for from about 1 dayto 2 days, from about 1 day to 3 days, from about 1 day to about 4 days,from about 1 day to about 5 days, from about 1 day to 6 days, from about1 day to 7 days, from about 1 day to 8 days, from about 1 day to 9 days,from about 1 day to 10 days, from about 2 days to 3 days, from about 2days to 4 days, from about 2 days to 5 days, from about 2 days to 6days, from about 2 days to 7 days, from about 2 days to 8 days, fromabout 2 days to 9 days, from about 2 days to 10 days, from about 3 daysto 4 days, from about 3 days to 5 days, from about 3 days to 6 days,from about 3 days to 7 days, from about 3 days to 8 days, from about 3days to 9 days, from about 3 days to 10 days, from about 4 days to 5days, from about 4 days to 6 days, from about 4 days to 7 days, fromabout 4 days to 8 days, from about 4 days to 9 days, from about 4 daysto 10 days, from about 5 days to 6 days, from about 5 days to 7 days,from about 5 days to 8 days, from about 5 days to 9 days, or from about5 days to 10 days.

In another aspect, the harvesting may be performed after the activatingwithin from about 4 days to about 12 days, from about 4 days to about 11days, from about 4 days to about 10 days, from about 4 days to about 9days, from about 4 days to about 8 days, from about 4 days to about 7days, from about 4 days to about 6 days, from about 4 days to about 5days, from about 5 days to about 12 days, from about 5 days to about 11days, from about 5 days to about 10 days, from about 5 days to about 9days, from about 5 days to about 8 days, from about 5 days to about 7days, or from about 5 days to about 6 days.

In another aspect, the number of the harvested T cells may be selectedfrom the group consisting of from about 2×10⁹ to about 5×10⁹, about5×10⁹ to about 10×10⁹, about 10×10⁹ to about 15×10⁹, about 5×10⁹ toabout 35×10⁹, about 5×10⁹ to about 30×10⁹, about 10×10⁹ to about 30×10⁹,about 15×10⁹ to about 20×10⁹, about 20×10⁹ to about 35×10⁹, about 24×10⁹to about 33×10⁹, and about 24.8×10⁹ to about 32.2×10⁹.

In another aspect, the activating, the transducing, the expanding, andthe harvesting may be performed in a closed or semi-closed system.

In another aspect, the closed system may be CliniMACS, Prodigy™, WAVE(XURI™) Bioreactor, WAVE (XURI™) Bioreactor in combination with BioSafeSepax™ II, G-Rex/GatheRex™ closed system, or G-Rex/GatheRex™ closedsystem in combination with BioSafe Sepax™ II.

BRIEF DESCRIPTION OF THE DRAWINGS

For a further understanding of the nature, objects, and advantages ofthe present disclosure, reference should be had to the followingdetailed description, read in conjunction with the following drawings,wherein like reference numerals denote like elements.

FIGS. 1A and 1B show loss of T_(naive/scm) and T_(cm) phenotype byprolonging ex vivo culturing of T cells obtained from different donors.

FIG. 2 shows reduction of IFN-γ secretion in cells grown on Day 15 ascompared with that grown on Day 10 from different donors.

FIG. 3 shows an experimental design to test the effect of restingconditions on T cell activation and expansion.

FIG. 4 shows CD25, CD69, and hLDL-R expression levels in differentexperimental groups.

FIGS. 5A and 5B show fold expansion and cell viability in differentexperimental groups on Day 7 expansion and Day 10 expansion,respectively.

FIG. 6 shows fold expansion and viability of activated T cellstransduced with a viral vector in different experimental groups on Day9.

FIG. 7 shows fold expansion and viability of activated T cellstransduced with a viral vector in different experimental groups on Day9.

FIG. 8 shows transgene expression in T cells resulting from differentresting time and in different scale production.

FIG. 9 shows fold expansion on Day 10 resulting from different restingtime and in different scale production.

FIG. 10 shows an experimental design to test the effect of concentrationof anti-CD3 and anti-CD28 antibodies on T cell activation.

FIG. 11 shows CD25, CD69, and hLDL-R expression in T cells activated bydifferent concentrations of anti-CD3 and anti-CD28 antibodies.

FIG. 12 shows, on Day 10 expansion, cell counts of T cells activated bydifferent concentrations of anti-CD3 and anti-CD28 antibodies in thepresence of different concentrations of IL-15.

FIG. 13 shows tetramer staining of recombinant TCR-transduced T cellsactivated by different concentrations of anti-CD3 and anti-CD28antibodies in the presence of different concentrations of IL-15.

FIG. 14A shows the percentage of CD3⁺CD8⁺Tetramer⁺ T cells resultingfrom different durations of activation.

FIG. 14B shows transgene expression resulting from different durationsof activation.

FIG. 15 shows CD25, CD69, and LDL-R expression in T cells activated byplate-bound or flask-bound anti-CD3 and anti-CD28 antibodies.

FIG. 16A shows levels of transduction in flask-bound (FB) andplate-bound (PB) activated T cells.

FIG. 16B shows fold expansion in flask-bound (FB) and plate-bound (PB)activated T cells.

FIG. 17 shows antigen specific IFN-γ levels elicited by flask-bound (FB)activated LV-R73 (a lentiviral vector expressing a T cell receptor)transduced T cells and plate-bound (PB) activated transduced T cells inresponse to tumor cells expressing a tumor associated antigen (TAA) indifferent donors.

FIG. 18 shows an experimental design to test the effect of using bagsand plates coated with anti-CD3 and anti-CD28 antibodies on T cellactivation.

FIG. 19 shows CD25, CD69, and LDL-R expression in T cells activated inbag-bound or flask-bound anti-CD3 and anti-CD28 antibodies.

FIG. 20 shows, on Day 6 expansion, cell expansion resulting from T cellsactivated by bag-bound antibodies at different concentrations and thatof T cells activated under FB conditions.

FIG. 21 shows, on Day 10 expansion, cell expansion resulting from Tcells activated by bag-bound antibodies at different concentrations andthat of T cells activated under FB conditions.

FIG. 22 shows a T cell manufacturing process according to one embodimentof the present disclosure.

FIG. 23A shows fold expansion of T cells manufactured according to oneembodiment of the present disclosure.

FIG. 23B shows transduced TCR expression of T cells manufacturedaccording to one embodiment of the present disclosure.

FIG. 23C shows phenotypes of T cells manufactured according to oneembodiment of the present disclosure.

FIG. 23D shows tumor cell growth inhibitory activity of T cellsmanufactured according to one embodiment of the present disclosure.

FIG. 23E shows tumor cell growth inhibitory activity of T cellsmanufactured according to another embodiment of the present disclosure.

FIG. 23F shows tumor cell growth inhibitory activity of T cellsmanufactured according to another embodiment of the present disclosure.

FIG. 23G shows tumor cell killing activity of T cells manufacturedaccording to another embodiment of the present disclosure.

FIG. 23H shows tumor cell killing activity of T cells manufacturedaccording to another embodiment of the present disclosure.

FIG. 24 shows T cell manufacturing process with overnight rest (about 16hours).

FIG. 25A shows fold expansion of T cells manufactured with overnightrest (about 16 hours).

FIG. 25B shows transduced TCR expression of T cells manufactured withovernight rest (about 16 hours).

FIG. 25C shows phenotypes of T cells manufactured with overnight rest(about 16 hours).

FIG. 25D shows tumor cell growth inhibitory activity of T cellsmanufactured with overnight rest (about 16 hours).

FIGS. 25E and 25F show cytotoxic activity of T cells manufactured withovernight rest (about 16 hours).

FIG. 26 shows ex vivo manipulation protocol in open and closed systems.

FIG. 27 shows ex vivo manipulation protocol in closed system inaccordance with one embodiment of the present disclosure.

FIG. 28 shows ex vivo manipulation protocol in closed system inaccordance with another embodiment of the present disclosure.

FIG. 29 shows IFN-γ release from T cells manufactured in open and closedsystems.

FIG. 30 shows a schematic of T cell manufacturing in accordance withsome embodiments of the present disclosure.

FIG. 31 shows a representative turnaround time from leukapheresiscollection to infusion-ready in accordance with one embodiment of thepresent disclosure. LP^(#):Leukapheresis collection, processing & freeze(optional). CoA: Additional time required for issuance of Certificate ofAnalysis.

FIG. 32 shows a T cell manufacturing process in accordance with oneembodiment of the present disclosure.

FIG. 33 shows T cell memory phenotyping of T cells produced by amanufacturing process in accordance with one embodiment of the presentdisclosure.

FIG. 34 shows CD27 and CD28 co-stimulation phenotyping of T cellsproduced by a manufacturing process in accordance with one embodiment ofthe present disclosure.

FIG. 35 shows T cell growth induced by IL-7, IL-15, or IL-2 decreases inan expansion time-dependent manner in accordance with one embodiment ofthe present disclosure.

FIG. 36 shows IFN-γ secretion decreases in an expansion time-dependentmanner in accordance with one embodiment of the present disclosure.

FIG. 37 shows EC₅₀ increases in an expansion time-dependent manner inaccordance with one embodiment of the present disclosure.

FIG. 38 shows expansion metrics in accordance with one embodiment of thepresent disclosure.

FIG. 39 shows surface expression of TCR in accordance with oneembodiment of the present disclosure.

FIG. 40 shows T-cell memory phenotype of the final products inaccordance with one embodiment of the present disclosure.

FIG. 41 shows IFN-γ release in response to exposure to target cells inaccordance with one embodiment of the present disclosure.

FIG. 42 shows EC₅₀ determination in accordance with one embodiment ofthe present disclosure.

FIG. 43 shows cytotoxic potential of T cells in accordance with oneembodiment of the present disclosure.

FIG. 44 shows a comparison in cell recovery between T cell productsobtained from healthy donors and cancer patients in accordance with anembodiment of the present disclosure.

FIG. 45 shows a comparison in cell viability between T cell productsobtained from healthy donors and cancer patients in accordance with anembodiment of the present disclosure.

FIG. 46 shows a comparison in fold expansion between T cell productsobtained from healthy donors and cancer patients in accordance with anembodiment of the present disclosure.

FIG. 47 shows a comparison in cell phenotype between T cell productsobtained from healthy donors and cancer patients in accordance with anembodiment of the present disclosure.

FIG. 48 shows a comparison in cell phenotype between T cell productsobtained from healthy donors and cancer patients in accordance with anembodiment of the present disclosure.

FIG. 49 shows TCR expression of T cell products in accordance with anembodiment of the present disclosure.

FIG. 50 shows a comparison in TCR expression between T cell productsobtained from healthy donors and cancer patients in accordance with anembodiment of the present disclosure.

FIG. 51 shows a comparison in TCR expression between T cell productsobtained from healthy donors and cancer patients in accordance with anembodiment of the present disclosure.

FIG. 52 shows gating scheme and T_(memory) subsets in accordance with anembodiment of the present disclosure.

FIG. 53 shows a comparison in cell phenotype between T cell productsobtained from healthy donors and cancer patients in accordance with anembodiment of the present disclosure.

FIG. 54 shows cytokine expression in T cell products in accordance withan embodiment of the present disclosure.

FIG. 55 shows cytokine expression in T cell products obtained fromhealthy donor in accordance with an embodiment of the presentdisclosure.

FIG. 56 shows a comparison in cytokine expression between T cellproducts obtained from healthy donors and cancer patients in accordancewith an embodiment of the present disclosure.

FIG. 57 shows IFN-γ release from T cell products obtained from cancerpatients in accordance with an embodiment of the present disclosure.

FIG. 58 shows IFN-γ release from T cell products obtained from healthydonors in accordance with an embodiment of the present disclosure.

FIG. 59 shows IFN-γ release from T cell products obtained from healthydonors in accordance with an embodiment of the present disclosure.

FIG. 60 shows IFN-γ release from T cell products obtained from cancerpatients in accordance with an embodiment of the present disclosure.

FIG. 61 shows cell killing activity of T cell products obtained fromhealthy donors in accordance with an embodiment of the presentdisclosure.

FIG. 62 shows cell killing activity of T cell products obtained fromhealthy donors in accordance with an embodiment of the presentdisclosure.

FIG. 63A shows a comparison in cell killing between T cell productsobtained from healthy donors and cancer patients in accordance with anembodiment of the present disclosure.

FIG. 63B shows a comparison in cell killing between T cell productsobtained from healthy donors and cancer patients in accordance with anembodiment of the present disclosure.

FIG. 63C shows a comparison in cell killing between T cell productsobtained from healthy donors and cancer patients in accordance with anembodiment of the present disclosure.

DETAILED DESCRIPTION

In an aspect, the disclosure provides for T cells populations producedby a method including thawing frozen peripheral blood mononuclear cells(PBMC), resting the thawed PBMC, activating the T cell in the restedPBMC with an anti-CD3 antibody and an anti-CD28 antibody immobilized ona solid phase, expanding the activated T cell, and obtaining the T cellpopulation comprising the expanded T cell.

In an aspect, the disclosure provides for methods of transducing a Tcell including thawing frozen peripheral blood mononuclear cells (PBMC),resting the thawed PBMC, activating the T cell in the cultured PBMC withan anti-CD3 antibody and an anti-CD28 antibody, transducing theactivated T cell with a viral vector, expanding the transduced T cell,and obtaining the expanded T cells; method of preparing a T cellpopulation, including thawing frozen peripheral blood mononuclear cells(PBMC), resting the thawed PBMC, activating the T cell in the restedPBMC with an anti-CD3 antibody and an anti-CD28 antibody immobilized ona solid phase, expanding the activated T cell, and obtaining the T cellpopulation comprising the expanded T cell; and methods of treating apatient or individual having a cancer or in need of a treatment thereof,comprising administering to the patient an effective amount of theexpanded T cells described herein. In an aspect, the patient orindividual in need thereof is a cancer patient. In an aspect, the cancerto be treated is selected from one or more of hepatocellular carcinoma(HCC), colorectal carcinoma (CRC), glioblastoma (GB), gastric cancer(GC), esophageal cancer, non-small cell lung cancer (NSCLC), pancreaticcancer (PC), renal cell carcinoma (RCC), benign prostate hyperplasia(BPH), prostate cancer (PCA), ovarian cancer (OC), melanoma, breastcancer, chronic lymphocytic leukemia (CLL), Merkel cell carcinoma (MCC),small cell lung cancer (SCLC), Non-Hodgkin lymphoma (NHL), acute myeloidleukemia (AML), gallbladder cancer and cholangiocarcinoma (GBC, CCC),urinary bladder cancer (UBC), acute lymphocytic leukemia (ALL), anduterine cancer (UEC).

T-cell based immunotherapy targets peptide epitopes derived fromtumor-associated or tumor-specific proteins, which are presented bymolecules of the major histocompatibility complex (MHC). The antigensthat are recognized by the tumor specific T lymphocytes, that is, theepitopes thereof, can be molecules derived from all protein classes,such as enzymes, receptors, transcription factors, etc. which areexpressed and, as compared to unaltered cells of the same origin,usually up-regulated in cells of the respective tumor.

There are two classes of MHC-molecules, MHC class I and MHC class II.MHC class I molecules are composed of an alpha heavy chain andbeta-2-microglobulin, MHC class II molecules of an alpha and a betachain. Their three-dimensional conformation results in a binding groove,which is used for non-covalent interaction with peptides. MHC class Imolecules can be found on most nucleated cells. They present peptidesthat result from proteolytic cleavage of predominantly endogenousproteins, defective ribosomal products (DRIPs) and larger peptides.However, peptides derived from endosomal compartments or exogenoussources are also frequently found on MHC class I molecules. Thisnon-classical way of class I presentation is referred to ascross-presentation. MHC class II molecules can be found predominantly onprofessional antigen presenting cells (APCs), and primarily presentpeptides of exogenous or transmembrane proteins that are taken up byAPCs e.g., during endocytosis, and are subsequently processed.

Complexes of peptide and MHC class I are recognized by CD8-positiveT-cells bearing the appropriate T-cell receptor (TCR), whereas complexesof peptide and MHC class II molecules are recognized byCD4-positive-helper-T-cells bearing the appropriate TCR. It is wellknown that the TCR, the peptide and the MHC are thereby present in astoichiometric amount of 1:1:1.

CD4-positive helper T-cells play an important role in inducing andsustaining effective responses by CD8-positive cytotoxic T-cells. Theidentification of CD4-positive T-cell epitopes derived from tumorassociated antigens (TAA) is of great importance for the development ofpharmaceutical products for triggering anti-tumor immune responses. Atthe tumor site, T helper cells, support a cytotoxic T-cell-(CTL-)friendly cytokine milieu and attract effector cells, e.g., CTLs, naturalkiller (NK) cells, macrophages, and granulocytes.

In the absence of inflammation, expression of MHC class II molecules ismainly restricted to cells of the immune system, especially professionalantigen-presenting cells (APC), e.g., monocytes, monocyte-derived cells,macrophages, dendritic cells. In cancer patients, cells of the tumorhave been found to express MHC class II molecules. Elongated (longer)peptides of the description can function as MHC class II activeepitopes.

T-helper cells, activated by MHC class II epitopes, play an importantrole in orchestrating the effector function of CTLs in anti-tumorimmunity. T-helper cell epitopes that trigger a T-helper cell responseof the TH1 type support effector functions of CD8-positive killerT-cells, which include cytotoxic functions directed against tumor cellsdisplaying tumor-associated peptide/MHC complexes on their cellsurfaces. In this way tumor-associated T-helper cell peptide epitopes,alone or in combination with other tumor-associated peptides, can serveas active pharmaceutical ingredients of vaccine compositions thatstimulate anti-tumor immune responses.

It was shown in mammalian animal models, e.g., mice, that even in theabsence of CD8-positive T lymphocytes, CD4-positive T-cells aresufficient for inhibiting manifestation of tumors via inhibition ofangiogenesis by secretion of interferon-gamma (IFN-γ). There is evidencefor CD4-positive T-cells as direct anti-tumor effectors.

Since the constitutive expression of HLA class II molecules is usuallylimited to immune cells, the possibility of isolating class II peptidesdirectly from primary tumors was previously not considered possible.However, Dengjel et al. were successful in identifying a number of MHCClass II epitopes directly from tumors (WO 2007/028574, EP 1 760 088B1,the contents of which are herein incorporated by reference in theirentirety).

Since both types of response, CD8 and CD4 dependent, contribute jointlyand synergistically to the anti-tumor effect, the identification andcharacterization of tumor-associated antigens recognized by either CD8+T-cells (ligand: MHC class I molecule+peptide epitope) or byCD4-positive T-helper cells (ligand: MHC class II molecule+peptideepitope) is important in the development of tumor vaccines.

For an MHC class I peptide to trigger (elicit) a cellular immuneresponse, it also must bind to an MHC-molecule. This process isdependent on the allele of the MHC-molecule and specific polymorphismsof the amino acid sequence of the peptide. MHC-class-1-binding peptidesare usually 8-12 amino acid residues in length and usually contain twoconserved residues (“anchors”) in their sequence that interact with thecorresponding binding groove of the MHC-molecule. In this way, each MHCallele has a “binding motif” determining which peptides can bindspecifically to the binding groove.

In the MHC class I dependent immune reaction, peptides not only have tobe able to bind to certain MHC class I molecules expressed by tumorcells, they subsequently also have to be recognized by T-cells bearingspecific T-cell receptors (TCR).

For proteins to be recognized by T-lymphocytes as tumor-specific or-associated antigens, and to be used in a therapy, particularprerequisites must be fulfilled. The antigen should be expressed mainlyby tumor cells and not, or in comparably small amounts, by normalhealthy tissues. In a preferred embodiment, the peptide should beover-presented by tumor cells as compared to normal healthy tissues. Itis furthermore desirable that the respective antigen is not only presentin a type of tumor, but also in high concentrations (i.e., copy numbersof the respective peptide per cell). Tumor-specific and tumor-associatedantigens are often derived from proteins directly involved intransformation of a normal cell to a tumor cell due to their function,e.g., in cell cycle control or suppression of apoptosis. Additionally,downstream targets of the proteins directly causative for atransformation may be up-regulated and thus may be indirectlytumor-associated. Such indirect tumor-associated antigens may also betargets of a vaccination approach. Epitopes are present in the aminoacid sequence of the antigen, in order to ensure that such a peptide(“immunogenic peptide”), being derived from a tumor associated antigen,and leads to an in vitro or in vivo T-cell-response.

Therefore, TAAs are a starting point for the development of a T-cellbased therapy including but not limited to tumor vaccines. The methodsfor identifying and characterizing the TAAs are usually based on the useof T-cells that can be isolated from patients or healthy subjects, orthey are based on the generation of differential transcription profilesor differential peptide expression patterns between tumors and normaltissues. However, the identification of genes over-expressed in tumortissues or human tumor cell lines, or selectively expressed in suchtissues or cell lines, does not provide precise information as to theuse of the antigens being transcribed from these genes in an immunetherapy. This is because only an individual subpopulation of epitopes ofthese antigens are suitable for such an application since a T-cell witha corresponding TCR has to be present and the immunological tolerancefor this particular epitope needs to be absent or minimal. In a verypreferred embodiment of the description it is therefore important toselect only those over- or selectively presented peptides against whicha functional and/or a proliferating T-cell can be found. Such afunctional T-cell is defined as a T-cell, which upon stimulation with aspecific antigen can be clonally expanded and is able to executeeffector functions (“effector T-cell”).

The term “T-cell receptor (TCR)” as used herein refers to a proteinreceptor on T cells that is composed of a heterodimer of an alpha (α)and beta (β) chain, although in some cells the TCR consists of gamma anddelta (γ/δ) chains. In embodiments of the disclosure, the TCR may bemodified on any cell comprising a TCR, including a helper T cell, acytotoxic T cell, a memory T cell, regulatory T cell, natural killer Tcell, and gamma delta T cell, for example.

TCR is a molecule found on the surface of T lymphocytes (or T cells)that is generally responsible for recognizing antigens bound to majorhistocompatibility complex (MHC) molecules. It is a heterodimerconsisting of an alpha and beta chain in 95% of T cells, while 5% of Tcells have TCRs consisting of gamma and delta chains. Engagement of theTCR with antigen and MHC results in activation of its T lymphocytethrough a series of biochemical events mediated by associated enzymes,co-receptors, and specialized accessory molecules. In immunology, theCD3 antigen (CD stands for cluster of differentiation) is a proteincomplex composed of four distinct chains (CD3-γ, CD3δ, and two timesCD3ε) in mammals, that associate with molecules known as the T-cellreceptor (TCR) and the ζ-chain to generate an activation signal in Tlymphocytes. The TCR, ζ-chain, and CD3 molecules together comprise theTCR complex. The CD3-γ, CD3δ, and CD3ε chains are highly related cellsurface proteins of the immunoglobulin superfamily containing a singleextracellular immunoglobulin domain. The transmembrane region of the CD3chains is negatively charged, a characteristic that allows these chainsto associate with the positively charged TCR chains (TCRα and TCRβ). Theintracellular tails of the CD3 molecules contain a single conservedmotif known as an immunoreceptor tyrosine-based activation motif or ITAMfor short, which is essential for the signalling capacity of the TCR.

CD28 is one of the molecules expressed on T cells that provideco-stimulatory signals, which are required for T cell activation. CD28is the receptor for B7.1 (CD80) and B7.2 (CD86). When activated byToll-like receptor ligands, the B7.1 expression is upregulated inantigen presenting cells (APCs). The B7.2 expression on antigenpresenting cells is constitutive. CD28 is the only B7 receptorconstitutively expressed on naive T cells. Stimulation through CD28 inaddition to the TCR can provide a potent co-stimulatory signal to Tcells for the production of various interleukins (IL-2 and IL-6 inparticular).

In an aspect, expansion and/or activation of T cells take place in thepresence of one or more of IL-2, IL-7, IL-10, IL-12, IL-15, IL-21. Inanother aspect, expansion and/or activation of T cells takes place withIL-2 alone, IL-7 alone, IL-15 alone, a combination of IL-2 and IL-15, ora combination of IL-7 and IL-15.

TCR constructs of the present disclosure may be applicable in subjectshaving or suspected of having cancer by reducing the size of a tumor orpreventing the growth or re-growth of a tumor in these subjects.Accordingly, the present disclosure further relates to a method forreducing growth or preventing tumor formation in a subject byintroducing a TCR construct of the present disclosure into an isolated Tcell of the subject and reintroducing into the subject the transformed Tcell, thereby effecting anti-tumor responses to reduce or eliminatetumors in the subject. Suitable T cells that can be used includecytotoxic lymphocytes (CTL) or any cell having a T cell receptor in needof disruption. As is well-known to one of skill in the art, variousmethods are readily available for isolating these cells from a subject.For example, using cell surface marker expression or using commerciallyavailable kits (e.g., ISOCELL™ from Pierce, Rockford, Ill.).

It is contemplated that the TCR construct can be introduced into thesubject's own T cells as naked DNA or in a suitable vector. Methods ofstably transfecting T cells by electroporation using naked DNA in theart. See, e.g., U.S. Pat. No. 6,410,319, the content of which isincorporated by reference in its entirety. Naked DNA generally refers tothe DNA encoding a TCR of the present disclosure contained in a plasmidexpression vector in proper orientation for expression. Advantageously,the use of naked DNA reduces the time required to produce T cellsexpressing the TCR of the present disclosure.

Alternatively, a viral vector (e.g., a retroviral vector, adenoviralvector, adeno-associated viral vector, or lentiviral vector) can be usedto introduce the TCR construct into T cells. Suitable vectors for use inaccordance with the method of the present disclosure are non-replicatingin the subject's T cells. A large number of vectors are known that arebased on viruses, where the copy number of the virus maintained in thecell is low enough to maintain the viability of the cell. Illustrativevectors include the pFB-neo vectors (STRATAGENE®) as well as vectorsbased on HIV, SV40, EBV, HSV, or BPV.

Once it is established that the transfected or transduced T cell iscapable of expressing the TCR construct as a surface membrane proteinwith the desired regulation and at a desired level, it can be determinedwhether the TCR is functional in the host cell to provide for thedesired signal induction. Subsequently, the transduced T cells arereintroduced or administered to the subject to activate anti-tumorresponses in the subject.

To facilitate administration, the transduced T cells according to thedisclosure can be made into a pharmaceutical composition or made into animplant appropriate for administration in vivo, with appropriatecarriers or diluents, which further can be pharmaceutically acceptable.The means of making such a composition or an implant have been describedin the art (see, for instance, Remington's Pharmaceutical Sciences, 16thEd., Mack, ed. (1980, the content which is herein incorporated byreference in its entirety)). Where appropriate, the transduced T cellscan be formulated into a preparation in semisolid or liquid form, suchas a capsule, solution, injection, inhalant, or aerosol, in the usualways for their respective route of administration. Means known in theart can be utilized to prevent or minimize release and absorption of thecomposition until it reaches the target tissue or organ, or to ensuretimed-release of the composition. Desirably, however, a pharmaceuticallyacceptable form is employed that does not hinder the cells fromexpressing the TCR. Thus, desirably the transduced T cells can be madeinto a pharmaceutical composition containing a balanced salt solution,preferably Hanks' balanced salt solution, or normal saline.

In certain aspects, the invention includes a method of making and/orexpanding the antigen-specific redirected T cells that comprisestransfecting T cells with an expression vector containing a DNAconstruct encoding TCR, then, optionally, stimulating the cells withantigen positive cells, recombinant antigen, or an antibody to thereceptor to cause the cells to proliferate.

In another aspect, a method is provided of stably transfecting andre-directing T cells by electroporation, or other non-viral genetransfer (such as, but not limited to sonoporation) using naked DNA.Most investigators have used viral vectors to carry heterologous genesinto T cells. By using naked DNA, the time required to produceredirected T cells can be reduced. “Naked DNA” means DNA encoding a TCRcontained in an expression cassette or vector in proper orientation forexpression. The electroporation method of this disclosure producesstable transfectants that express and carry on their surfaces the TCR.

In certain aspects, the T cells are primary human T cells, such as Tcells derived from human peripheral blood mononuclear cells (PBMC), PBMCcollected after stimulation with G-CSF, bone marrow, or umbilical cordblood. Conditions include the use of mRNA and DNA and electroporation.Following transfection, cells may be immediately infused or may bestored. In certain aspects, following transfection, the cells may bepropagated for days, weeks, or months ex vivo as a bulk populationwithin about 1, 2, 3, 4, 5 days or more following gene transfer intocells. In a further aspect, following transfection, the transfectantsare cloned and a clone demonstrating presence of a single integrated orepisomally maintained expression cassette or plasmid, and expression ofthe TCR is expanded ex vivo. The clone selected for expansiondemonstrates the capacity to specifically recognize and lysepeptide-expressing target cells. The recombinant T cells may be expandedby stimulation with IL-2, or other cytokines that bind the commongamma-chain (e.g., IL-7, IL-12, IL-15, IL-21, and others). Therecombinant T cells may be expanded by stimulation with artificialantigen presenting cells. The recombinant T cells may be expanded onartificial antigen presenting cell or with an antibody, such as OKT3,which cross links CD3 on the T cell surface. Subsets of the recombinantT cells may be deleted on artificial antigen presenting cell or with anantibody, such as Campath, which binds CD52 on the T cell surface. In afurther aspect, the genetically modified cells may be cryopreserved.

A composition of the present invention can be provided in unit dosageform wherein each dosage unit, e.g., an injection, contains apredetermined amount of the composition, alone or in appropriatecombination with other active agents. The term unit dosage form as usedherein refers to physically discrete units suitable as unitary dosagesfor human and animal subjects, each unit containing a predeterminedquantity of the composition of the present invention, alone or incombination with other active agents, calculated in an amount sufficientto produce the desired effect, in association with a pharmaceuticallyacceptable diluent, carrier, or vehicle, where appropriate. Thespecifications for the novel unit dosage forms of the present inventiondepend on the particular pharmacodynamics associated with thepharmaceutical composition in the particular subject.

Desirably an effective amount or sufficient number of the isolatedtransduced T cells is present in the composition and introduced into thesubject such that long-term, specific, anti-tumor responses areestablished to reduce the size of a tumor or eliminate tumor growth orregrowth than would otherwise result in the absence of such treatment.Desirably, the amount of transduced T cells reintroduced into thesubject causes an about 10%, about 20%, about 30%, about 40%, about 50%,about 60%, about 70%, about 80%, about 90%, about 95%, about 98%, orabout 99% decrease in tumor size when compared to otherwise sameconditions wherein the transduced T cells are not present.

Accordingly, the amount of transduced T cells administered should takeinto account the route of administration and should be such that asufficient number of the transduced T cells will be introduced so as toachieve the desired therapeutic response. Furthermore, the amounts ofeach active agent included in the compositions described herein (e.g.,the amount per each cell to be contacted or the amount per certain bodyweight) can vary in different applications. In general, theconcentration of transduced T cells desirably should be sufficient toprovide in the subject being treated at least from about 1×10⁶ to about1×10⁹ transduced T cells/m² (or kg) of a patient, even more desirably,from about 1×10⁷ to about 5×10⁶ transduced T cells/m² (or kg) of apatient, although any suitable amount can be utilized either above,e.g., greater than 5×10⁶ cells/m² (or kg) of a patient, or below, e.g.,less than 1×10⁷ cells/m² (or kg) of a patient. The dosing schedule canbe based on well-established cell-based therapies (see, e.g., U.S. Pat.No. 4,690,915, the content which is herein incorporated by reference inits entirety), or an alternate continuous infusion strategy can beemployed.

These values provide general guidance of the range of transduced T cellsto be utilized by the practitioner upon optimizing the method of thepresent invention for practice of the invention. The recitation hereinof such ranges by no means precludes the use of a higher or lower amountof a component, as might be warranted in a particular application. Forexample, the actual dose and schedule can vary depending on whether thecompositions are administered in combination with other pharmaceuticalcompositions, or depending on interindividual differences inpharmacokinetics, drug disposition, and metabolism. One skilled in theart readily can make any necessary adjustments in accordance with theexigencies of the particular situation.

The terms “T cell” or “T lymphocyte” are art-recognized and are intendedto include thymocytes, naïve T lymphocytes, immature T lymphocytes,mature T lymphocytes, resting T lymphocytes, or activated T lymphocytes.Illustrative populations of T cells suitable for use in particularembodiments include, but are not limited to, helper T cells (HTL; CD4+ Tcell), a cytotoxic T cell (CTL; CD8+ T cell), CD4+CD8+ T cell, CD4−CD8−T cell, or any other subset of T cells. Other illustrative populationsof T cells suitable for use in particular embodiments include, but arenot limited to, T cells expressing one or more of the following markers:CD3, CD4, CD8, CD27, CD28, CD45RA, CD45RO, CD62L, CD127, CD197, andHLA-DR and if desired, can be further isolated by positive or negativeselection techniques.

A peripheral blood mononuclear cell (PBMC) is defined as any blood cellwith a round nucleus (i.e., a lymphocyte, a monocyte, or a macrophage).These blood cells are a critical component in the immune system to fightinfection and adapt to intruders. The lymphocyte population consists ofCD4+ and CD8+ T cells, B cells and Natural Killer cells, CD14+monocytes, and basophils/neutrophils/eosinophils/dendritic cells. Thesecells are often separated from whole blood or from leukapheresisproducts using FICOLL™, a hydrophilic polysaccharide that separateslayers of blood, with monocytes and lymphocytes forming a buffy coatunder a layer of plasma. In one embodiment, “PBMCs” refers to apopulation of cells comprising at least T cells, and optionally NKcells, and antigen presenting cells.

The term “activation” refers to the state of a T cell that has beensufficiently stimulated to induce detectable cellular proliferation. Inparticular embodiments, activation can also be associated with inducedcytokine production, and detectable effector functions. The term“activated T cells” refers to, among other things, T cells that areproliferating. Signals generated through the TCR alone are insufficientfor full activation of the T cell and one or more secondary orcostimulatory signals are also required. Thus, T cell activationcomprises a primary stimulation signal through the TCR/CD3 complex andone or more secondary costimulatory signals. Co-stimulation can beevidenced by proliferation and/or cytokine production by T cells thathave received a primary activation signal, such as stimulation throughthe CD3/TCR complex or through CD2.

As used herein, a resting T cell means a T cell that is not dividing orproducing cytokines. Resting T cells are small (approximately 6-8microns) in size compared to activated T cells (approximately 12-15microns).

As used herein, a primed T cell is a resting T cell that has beenpreviously activated at least once and has been removed from theactivation stimulus for at least about 1 hour, at least about 2 hours,at least about 3 hours, at least about 4 hours, at least about 5 hours,at least about 6 hours, at least about 12 hours, at least about 24hours, at least about 48 hours, at least about 60 hours, at least about72 hours, at least about 84 hours, at least about 96 hours, at leastabout 108 hours, or at least about 120 hours. Alternatively, resting maybe carried out within a period of from about 0.5 hour to about 120hours, about 0.5 hour to about 108 hours, about 0.5 hour to about 96hours, about 0.5 hour to about 84 hours, about 0.5 hour to about 72hours, about 0.5 hour to about 60 hours, about 0.5 hour to about 48hours, about 0.5 hour to about 36 hours, about 0.5 hour to about 24hours, about 0.5 hour to about 18 hours, about 0.5 hour to about 12hours, about 0.5 hour to about 6 hours, about 1 hour to about 6 hours,about 2 hours to about 5 hours, about 3 hours to about 5 hours, or about4 hours to about 5 hours. Primed T cells usually have a memoryphenotype.

A population of T cells may be induced to proliferate by activating Tcells and stimulating an accessory molecule on the surface of T cellswith a ligand, which binds the accessory molecule. Activation of apopulation of T cells may be accomplished by contacting T cells with afirst agent which stimulates a TCR/CD3 complex-associated signal in theT cells. Stimulation of the TCR/CD3 complex-associated signal in a Tcell may be accomplished either by ligation of the T cell receptor(TCR)/CD3 complex or the CD2 surface protein, or by directly stimulatingreceptor-coupled signalling pathways. Thus, an anti-CD3 antibody, ananti-CD2 antibody, or a protein kinase C activator in conjunction with acalcium ionophore may be used to activate a population of T cells.

To induce proliferation, an activated population of T cells may becontacted with a second agent, which stimulates an accessory molecule onthe surface of the T cells. For example, a population of CD4+ T cellscan be stimulated to proliferate with an anti-CD28 antibody directed tothe CD28 molecule on the surface of the T cells. Alternatively, CD4+ Tcells can be stimulated with a natural ligand for CD28, such as B7-1 andB7-2. The natural ligand can be soluble, on a cell membrane, or coupledto a solid phase surface. Proliferation of a population of CD8+ T cellsmay be accomplished by use of a monoclonal antibody ES5.2D8, which bindsto CD9, an accessory molecule having a molecular weight of about 27 kDpresent on activated T cells. Alternatively, proliferation of anactivated population of T cells can be induced by stimulation of one ormore intracellular signals, which result from ligation of an accessorymolecule, such as CD28.

The agent providing the primary activation signal and the agentproviding the costimulatory agent can be added either in soluble form orcoupled to a solid phase surface. In a preferred embodiment, the twoagents may be coupled to the same solid phase surface.

Following activation and stimulation of an accessory molecule on thesurface of the T cells, the progress of proliferation of the T cells inresponse to continuing exposure to the ligand or other agent, which actsintracellularly to simulate a pathway mediated by the accessorymolecule, may be monitored. When the rate of T cell proliferationdecreases, T cells may be reactivated and re-stimulated, such as withadditional anti-CD3 antibody and a co-stimulatory ligand, to inducefurther proliferation. In one embodiment, the rate of T cellproliferation may be monitored by examining cell size. Alternatively, Tcell proliferation may be monitored by assaying for expression of cellsurface molecules in response to exposure to the ligand or other agent,such as B7-1 or B7-2. The monitoring and re-stimulation of T cells canbe repeated for sustained proliferation to produce a population of Tcells increased in number from about 100- to about 100,000-fold over theoriginal T cell population.

The method of the present disclosure can be used to expand selected Tcell populations for use in treating an infectious disease or cancer.The resulting T cell population can be genetically transduced and usedfor immunotherapy or can be used for in vitro analysis of infectiousagents. Following expansion of the T cell population to sufficientnumbers, the expanded T cells may be restored to the individual. Themethod of the present disclosure may also provide a renewable source ofT cells. Thus, T cells from an individual can be expanded ex vivo, aportion of the expanded population can be re-administered to theindividual and another portion can be frozen in aliquots for long termpreservation, and subsequent expansion and administration to theindividual. Similarly, a population of tumor-infiltrating lymphocytescan be obtained from an individual afflicted with cancer and the T cellsstimulated to proliferate to sufficient numbers and restored to theindividual.

The present disclosure may also pertain to compositions containing anagent that provides a costimulatory signal to a T cell for T cellexpansion (e.g., an anti-CD28 antibody, B7-1 or B7-2 ligand), coupled toa solid phase surface which may additionally include an agent thatprovides a primary activation signal to the T cell (e.g., an anti-CD3antibody) coupled to the same solid phase surface. These agents may bepreferably attached to beads or flasks or bags. Compositions comprisingeach agent coupled to different solid phase surfaces (i.e., an agentthat provides a primary T cell activation signal coupled to a firstsolid phase surface and an agent that provides a costimulatory signalcoupled to a second solid phase surface) may also be within the scope ofthis disclosure.

In an aspect, TAA peptides that are capable of use with the methods andembodiments described herein include, for example, those TAA peptidesdescribed in U.S. Publication 20160187351, U.S. Publication 20170165335,U.S. Publication 20170035807, U.S. Publication 20160280759, U.S.Publication 20160287687, U.S. Publication 20160346371, U.S. Publication20160368965, U.S. Publication 20170022251, U.S. Publication 20170002055,U.S. Publication 20170029486, U.S. Publication 20170037089, U.S.Publication 20170136108, U.S. Publication 20170101473, U.S. Publication20170096461, U.S. Publication 20170165337, U.S. Publication 20170189505,U.S. Publication 20170173132, U.S. Publication 20170296640, U.S.Publication 20170253633, U.S. Publication 20170260249, U.S. Publication20180051080, and U.S. Publication No. 20180164315, the contents of eachof these publications and sequence listings described therein are hereinincorporated by reference in their entireties.

In an aspect, T cells described herein selectively recognize cells whichpresent a TAA peptide described in one of more of the patents andpublications described above.

In another aspect, TAA that are capable of use with the methods andembodiments described herein include at least one selected from SEQ IDNO: 1 to SEQ ID NO: 157.

SEQ ID Amino Acid NO: Sequence   1 YLYDSETKNA   2 HLMDQPLSV   3GLLKKINSV   4 FLVDGSSAL   5 FLFDGSANLV   6 FLYKIIDEL   7 FILDSAETTTL   8SVDVSPPKV   9 VADKIHSV  10 IVDDLTINL  11 GLLEELVTV  12 TLDGAAVNQV  13SVLEKEIYSI  14 LLDPKTIFL  15 YTFSGDVQL  16 YLMDDFSSL  17 KVWSDVTPL  18LLWGHPRVALA  19 KIWEELSVLEV  20 LLIPFTIFM  21 FLIENLLAA  22 LLWGHPRVALA 23 FLLEREQLL  24 SLAETIFIV  25 TLLEGISRA  26 ILQDGQFLV  27 VIFEGEPMYL 28 SLFESLEYL  29 SLLNQPKAV  30 GLAEFQENV  31 KLLAVIHEL  32 TLHDQVHLL 33 TLYNPERTITV  34 KLQEKIQEL  35 SVLEKEIYSI  36 RVIDDSLVVGV  37VLFGELPAL  38 GLVDIMVHL  39 FLNAIETAL  40 ALLQALMEL  41 ALSSSQAEV  42SLITGQDLLSV  43 QLIEKNWLL  44 LLDPKTIFL  45 RLHDENILL  46 YTFSGDVQL  47GLPSATTTV  48 GLLPSAESIKL  49 KTASINQNV  50 SLLQHLIGL  51 YLMDDFSSL  52LMYPYIYHV  53 KVWSDVTPL  54 LLWGHPRVALA  55 VLDGKVAVV  56 GLLGKVTSV  57KMISAIPTL  58 GLLETTGLLAT  59 TLNTLDINL  60 VIIKGLEEI  61 YLEDGFAYV  62KIWEELSVLEV  63 LLIPFTIFM  64 ISLDEVAVSL  65 KISDFGLATV  66 KLIGNIHGNEV 67 ILLSVLHQL  68 LDSEALLTL  69 VLQENSSDYQSNL  70 HLLGEGAFAQV  71SLVENIHVL  72 YTFSGDVQL  73 SLSEKSPEV  74 AMFPDTIPRV  75 FLIENLLAA  76FTAEFLEKV  77 ALYGNVQQV  78 LFQSRIAGV  79 ILAEEPIYIRV  80 FLLEREQLL  81LLLPLELSLA  82 SLAETIFIV  83 AILNVDEKNQV  84 RLFEEVLGV  85 YLDEVAFML  86KLIDEDEPLFL  87 KLFEKSTGL  88 SLLEVNEASSV  89 GVYDGREHTV  90 GLYPVTLVGV 91 ALLSSVAEA  92 TLLEGISRA  93 SLIEESEEL  94 ALYVQAPTV  95 KLIYKDLVSV 96 ILQDGQFLV  97 SLLDYEVSI  98 LLGDSSFFL  99 VIFEGEPMYL 100 ALSYILPYL101 FLFVDPELV 102 SEWGSPHAAVP 103 ALSELERVL 104 SLFESLEYL 105 KVLEYVIKV106 VLLNEILEQV 107 SLLNQPKAV 108 KMSELQTYV 109 ALLEQTGDMSL 110VIIKGLEEITV 111 KQFEGTVEI 112 KLQEEIPVL 113 GLAEFQENV 114 NVAEIVIHI 115ALAGIVTNV 116 NLLIDDKGTIKL 117 VLMQDSRLYL 118 KVLEHVVRV 119 LLWGNLPEI120 SLMEKNQSL 121 KLLAVIHEL 122 ALGDKFLLRV 123 FLMKNSDLYGA 124KLIDHQGLYL 125 GPGIFPPPPPQP 126 ALNESLVEC 127 GLAALAVHL 128 LLLEAVWHL129 SIIEYLPTL 130 TLHDQVHLL 131 SLLMWITQC 132 FLLDKPQDLSI 133 YLLDMPLWYL134 GLLDCPIFL 135 VLIEYNFSI 136 TLYNPERTITV 137 AVPPPPSSV 138 KLQEELNKV139 KLMDPGSLPPL 140 ALIVSLPYL 141 FLLDGSANV 142 ALDPSGNQLI 143 ILIKHLVKV144 VLLDTILQL 145 HLIAEIHTA 146 SMNGGVFAV 147 MLAEKLLQA 148 YMLDIFHEV149 ALWLPTDSATV 150 GLASRILDA 151 SYVKVLHHL 152 VYLPKIPSW 153 NYEDHFPLL154 VYIAELEKI 155 VHFEDTGKTLLF 156 VLSPFILTL 157 HLLEGSVGV

EXAMPLES Example 1

Autologous T Cell Manufacturing Process

Adoptive cell transfer of purified naïve (T_(n)), stem cell memory(T_(scm)), and central memory (T_(cm)) T cell subsets causes superiortumor regression compared with transfer of the more-differentiatedeffector memory (T_(em)) and effector (T_(eff)) T cells. Traditionalmanufacturing process for an engineered T cell product may take 10-15days long. However, a process longer than about 12 days, e.g., 14 days,may result in reduced potency of the cells, e.g., fewer more-effectiveT_(n), T_(scm), and T_(cm) T cell subsets and more less-effective T_(em)and T_(eff) T cell subsets. For example, FIG. 1A shows prolonging exvivo culturing of T cells, e.g., 14 days, from two healthy donors, e.g.,donor 6 and donor 8, in which the desirable T_(cm) T cell subsets werereduced from that cultured for 0, 6, or 10 days. On the other hand, themore differentiated and less persistent T_(em) T cell subsets wereincreased from that cultured for 0, 6, or 10 days. FIG. 1B showsprolonging ex vivo culturing of T cells, e.g., 14 days, from threepatients, e.g., patient 864, patient 453, and patient 265, in which thedesirable T_(cm) T cell subsets were reduced from that cultured for 0,6, or 10 days. On the other hand, the more differentiated and lesspersistent T_(em) T cell subsets were increased from that cultured for0, 6, or 10 days.

Fewer more-effective T_(n), T_(scm), and T_(cm) T cell subsets mayresult in fewer effectively activated T cells that secret cytokines,e.g., interferon gamma (INF-γ). FIG. 2 shows reduced INF-γ secretion byperipheral blood mononuclear cells (PBMC) obtained from three healthydonors, e.g., donor 6 (D6), donor 7 (D7), and donor 8 (D8), activatedand cultured for 15 days as compared with that activated and culturedfor 10 days.

To shorten the manufacturing process, embodiments of the presentdisclosure include an about 7 to about 10-day process leading to themanufacturing of over 10 billion (10×10⁹) cells without the loss ofpotency. In addition, the concentrations of several raw materials may beoptimized to reduce the cost of good by 30%.

Effect of Eliminating or Modifying Resting Conditions in Autologous TCell Manufacturing Process on T Cell Activation

FIG. 3 shows an experimental design used to test the effect of restingconditions on T cell activation and expansion. Briefly, group Arepresents a first batch of PBMC that were thawed on Day 0, followed byresting without cytokines overnight (O/N), i.e., 24 hours, followed byactivating the rested PBMC with anti-CD3 and anti-CD28 antibodiesimmobilized on non-tissue culture treated plates. IL-7 is a homeostaticcytokine that promotes survival of T cells by preventing apoptosis. IL-7may be added to PBMC during resting. Groups B1-B3 represent a secondbatch of PBMC that were thawed on Day 1, followed by resting in thepresence of IL-7 (group B1) or in the presence of IL-7+IL-15 (group B2)or without cytokine (group B3) for 4-6 hours, followed by activating therested PBMC with anti-CD3 and anti-CD28 antibodies immobilized onnon-tissue culture treated plates. Group C represents a third batch ofPBMC that were thawed on Day 1 (without resting and without cytokine),followed by activating the thawed PBMC with anti-CD3 and anti-CD28antibodies immobilized on tissue culture plates. Cells may be harvestedand counted on Day 8-10, followed by activation panel analysis.

CD25 and CD69 are activation markers on the surface of cytokine- ormitogen-activated lymphocytes. The binding and entry of the VSV-Gpseudotyped lentiviral vectors, such as LV-R73, has been shown to bemediated by interaction of the VSV-G envelop protein with low densitylipoprotein receptor (LDL-R) on the host cells. Resting T cells do notexpress LDL-R, however activation with anti-CD3 and anti-CD28 antibodiesinduces LDL-R expression on T cells and permits efficient lentiviraltransduction. This suggests that kinetics of LDL-R expression regulatedby level of activation can impact transduction efficiency with VSV-Glentiviral vector.

FIG. 4 shows CD25, CD69, and hLDL-R expression levels among groups A,131-133, and C are comparable, indicating that the time for resting maybe shortened, e.g., from 24 hours to 4-6 hours, without significantlyreducing T cell activation.

Effect of Eliminating or Modifying Resting Conditions in Autologous TCell Manufacturing Process on T Cell Expansion

FIGS. 5A and 5B show fold expansion and cell viability in groups A andB1-B3 are comparable on Day 7 expansion and Day 10 expansion,respectively. Group C, which is without resting, however, has the leastfold expansion on Day 7 expansion (5-fold) (FIG. 5A) and Day 10expansion (16-fold) (FIG. 5B). These results suggest that the time forresting may be shortened, e.g., from 24 hours to 4-6 hours, withoutsignificantly reducing T cell expansion.

FIGS. 6 and 7 show fold expansion and viability of activated T cellstransduced with a viral vector expressing TCR, e.g., LV-R73, in 2donors, i.e., donor 1 (FIG. 6) or donor 2 (FIG. 7), in groups A, B1-133,and C on Day 9 expansion. Groups B1 and B2 show better cell expansionthan groups A, B3, and C, indicating that brief resting time, e.g., 5hours, in the presence of cytokines, e.g., IL-7 or IL-7+IL-15, mayincrease expansion of transduced T cells. Rep1 and Rep2 represent tworeplicates. These results support use of shortened resting time, e.g.,from 24 hours to 4-6 hours, in autologous T cell manufacturing processin the presence of cytokines, e.g., IL-7 and/or IL-15, withoutsignificantly reducing T cell expansion.

Effect of Eliminating or Modifying Resting Conditions in Autologous TCell Manufacturing Process on Transgene Expression in T Cells

Using peptide/MHC complex-loaded tetramers to detect T cells expressingtransduced TCR that specifically binds peptide/MHC complex, FIG. 8 showscomparable transgene expression, e.g., recombinant TCR expression, in Tcells rested for 4 hours (with IL-7) and 24h (without cytokine) in a T75tissue culture flask or 4 hours (with IL-7) in G-Rex 100 flask in alarge-scale production run for donor 13, donor 14, and donor 16. Theseresults suggest that the resting time may be shortened, e.g., from 24hours to 4-6 hours in a scale-up manufacturing process, withoutsignificantly reducing transgene expression in transduced T cells. Inaddition, use of one G-Rex 100 flask can simplify the resting processfurther by replacing multiple T75 flasks.

FIG. 9 shows comparable fold expansion on Day 10 expansion in T cellsrested for 4-6 hours, 6 hours, or 24 hours in small scale or large-scaleproduction for donor 13, donor 14, and donor 16. These results suggestthat the resting time may be shortened, e.g., from 24 hours to 4-6 hoursin a scale-up manufacturing process, without significantly impacting theexpansion of transduced T cells.

Effect of Concentration of Anti-CD3 and Anti-CD28 Antibodies inAutologous T Cell Manufacturing Process on T Cell Activation

Activation is an important step in autologous T cell manufacturingprocesses because both transduction efficiency and rate of expansionrely on T cell activation. Stimulation of T cells via engagement of CD3receptor and a co-receptor, such as CD28, using antibodies is a commonmethod of activating T cells. T cell activation serves as a preparatorystep for transduction with viral vectors, such as lentiviral vector.

FIG. 10 shows an experimental design used to test the effect ofconcentration of anti-CD3 and anti-CD28 antibodies on T cell activation.Briefly, on Day 0, PBMC were thawed and cultured or rested withoutactivation cytokines overnight or 24 hours. On Day 1, the rested PBMCwere activated by incubating them in two 24-well plates coated withdifferent concentrations, e.g., 0.1 μg/ml, 0.25 μg/ml, 0.5 μg/ml, 1.0μg/ml, of anti-CD3 and anti-CD28 antibodies in the presence ofIL-7+IL-15. On Day 2, the activated T cells were analyzed for CD25,CD69, and hLDL-R expression and transduced with VSV-G pseudotypedlentiviral vectors, e.g., 1×Eng LV-R73. On Day 6/7 and 9, analyses, suchas cell counts, viability, and fluorescence-activated cell sorting(FACS) with dextramers (Dex), which are multimers based on a dextranbackbone bearing multiple fluorescein and peptide/MHC complexes fordetecting T cells expressing recombinant TCR, were performed.

FIG. 11 shows, prior to viral transduction, T cells activated with 0.5μg/ml and 1.0 μg/ml of anti-CD3 and anti-CD28 antibodies have comparablelevels of CD25, CD69, and hLDL-R expression within each donor 16 anddonor 14. However, these expression levels are significantly higher thanthose from T cells activated with lower concentrations, e.g., 0.1 μg/mland 0.25 μg/ml, of anti-CD3 and anti-CD28 antibodies. These resultssuggest that the concentration of anti-CD3 and anti-CD28 antibodies maybe reduced, e.g., from 1.0 μg/ml to 0.5 μg/ml, without significantlyreducing T cell activation.

Effect of Concentration of Anti-CD3 and Anti-CD28 Antibodies andCytokines in Autologous T Cell Manufacturing Process on T Cell Expansion

FIG. 12 shows, on Day 10 expansion, cell counts of T cells activated by0.5 μg/ml or 1.0 μg/ml of anti-CD3 and anti-CD28 antibodies in thepresence of different concentrations, e.g., 25 ng/ml, 50 ng/ml, or 100ng/ml, of IL-15 are comparable within each donor 16, donor 13, and donor14. These results suggest that the concentration of anti-CD3 andanti-CD28 antibodies may be reduced, e.g., from 1.0 μg/ml to 0.5 μg/ml,and the concentration of IL-15 may be reduced, e.g., from 100 ng/ml to25 ng/ml, without significantly reducing T cell expansion.

FIG. 13 shows tetramer staining of recombinant TCR-transduced T cellsactivated by 0.5 μg/ml or 1.0 μg/ml of anti-CD3 and anti-CD28 antibodiesin the presence of different concentrations, e.g., 25 ng/ml, 50 ng/ml,or 100 ng/ml, of IL-15 are comparable within each donor 16, donor 13,and donor 14. These results suggest that the concentration of anti-CD3and anti-CD28 antibodies may be reduced, e.g., from 1.0 μg/ml to 0.5μg/ml, and the concentration of IL-15 may be reduced, e.g., from 100ng/ml to 25 ng/ml, without significantly reducing viral transduction ofT cells.

Together, these results suggest that (1) resting time after thawing PBMCcan be shortened, e.g., from 24 hours to 4-6 hours, withoutsignificantly reducing T cell activation, transgene expression, and Tcell expansion; and (2) concentrations of anti-CD3 and anti-CD28antibodies can be reduced, e.g., from 1.0 μg/ml to 0.5 μg/ml, andconcentrations of cytokines can be reduced, such as IL-15, e.g., from100 ng/ml to 25 ng/ml, without significantly reducing T cell activation,transgene expression, and T cell expansion.

Effect of duration of activation in autologous T cell manufacturingprocess on transduction efficiency with lentiviral vector

One of the major goals of developing an autologous T cell manufacturingprocess is to improve the rate of transduction achieved in primary humanT cells with the lentiviral construct encoding TCR. Unlike gammaretroviruses that can transduce only dividing cells, lentiviruses, intheory, can transduce both dividing and non-dividing cells. However,transducing resting T cells with lentiviruses have yielded poortransduction efficiencies. Activation of T cells has been shown tofacilitate their transduction with a lentivirus. Thus, stimulation of Tcells with anti-CD3 and anti-CD28 antibodies in immobilized, beads orsoluble form, has become a pre-requisite for performing lentiviraltransduction and is a standard part of manufacturing geneticallymodified T cells for adoptive cell therapy.

Because the T cell activation step plays a critical role in preparing Tcells for transduction, the effective duration of activation withanti-CD3 and anti-CD28 antibodies may need to be optimized.

To determine the optimal duration of activation, a time course studyevaluating the effect of different duration of activation ontransduction efficiency was performed. The results show that optimalwindow for transducing T cells may be after 16-24h of activation withanti-CD3 and anti-CD28 antibodies. Thus, time for T-cell activationprior to transduction may be reduced from 48h to 16-24h for all furtherprocess development and clinical manufacturing.

In another embodiment of the present disclosure, for fresh PBMC, i.e.,not frozen, resting may not be needed. Thus, fresh PBMC, withoutresting, may be activated by anti-CD3 antibody and anti-CD28 antibody,followed by viral vector transduction to obtain transduced T cells.

Although methods of transducing T cells may involve sequential steps ofactivating T cells in tissue culture, followed by transferring theactivated T cells to different tissue culture, in which transducingactivated T cells with viral vectors takes place, activating andtransducing steps, however, may be carried out concurrently. Forexample, while T cells are being activated by anti-CD3 and anti-CD28antibodies, transducing activated T cells may be carried outsimultaneously in the same culture. By doing so, the entire T celltransducing process, i.e., from providing PBMC to obtaining transduced Tcells, may be shortened to, for example, 3-4 days.

Example 2

Determine optimal duration of T cell activation for the improvement oftransduction efficiency with lentiviral construct

PBMC from healthy donors were activated using anti-CD3 and anti-CD28antibodies for different time intervals in preparation for transduction.Activated T cells from PBMC were treated with concentrated supernatantsgenerated using different lentiviral constructs expressing TAA targetingR7P1 D5 TCR. Transduced cells were expanded in the presence of IL-7 andIL-15. The products were compared based on R7P1 D5 TCR transgeneexpression as determined by flow cytometry using specificdextramer/tetramer.

Representative Materials and Methods

Supplies Manufacturer Catalog # TexMACS media Miltenyi Biotec130-097-196 Human AB Serum Gemini 100-512 PBS/EDTA Lonza BE02-017F IL-7Peprotech 200-07 IL-15 Peprotech 200-15 Anti-CD3 antibody Ebioscience16-0037-85 Anti-CD28 antibody Ebioscience 16-0289-85 24-well non-tissueCo-star 3738 culture plates G-Rex 24-well plate Wilson Wolf 80192M 15 mLConical Tube Falcon 352097 50 ml conical tube Corning 430290 5 mLserological Pipet BD 53300-421 10 mL serological Pipet BD 53300-523 25ml serological pipet BD 53300-567 1000 ul pipet tips Rainin 17007954 200ul pipet tips Rainin 17007961 20 pL pipet tips Rainin 17007957 1.5 mLMicrocentrifuge Fisher 02-681-5 Tube AOPI Staining Solution NexcelomCS2-0106 PBS without Mg and Ca Lonza 17-516F/24 96 well plate Corning3799 P-20 Micropipettor Rainin 17014382 P-200 Micropipettor Rainin17014391 P-1000 Micropipettor Rainin 17017382 Pipettaid Drummond 193970LT75 flasks BD Falcon BD353136 T25 flask Corning 430372 Benzonase SigmaE1014 Protamine sulfate McKesson 804514 Lentivirus Lentigen LV-R73, R78,R72, R22 Live/Dead Aqua dye Thermo Fisher L-34966 ABC Comp Beads ThermoFisher A10497 CD3-BV421 BD 562426 CD8-APC Biolegend 301014 TAATetramer-PE lmmatics N/A

Representative Methods

To compare different durations of T cell activation, representativeexperiments described herein were carried out following standardsmall-scale T cells generation process involving, for example, 4 steps:thaw/rest, activation, transduction and expansion.

Thaw and Rest

Frozen PBMC from healthy donors (n=3, D3, D4, D9) were thawed in warmTexMACS media supplemented with 5% human AB serum. Cells were treatedwith benzonase nuclease (50U/ml) for 15 minutes at 37° C., washed,counted, and put to overnight rest in complete TexMACS media.

Activation

On a day when cells are thawed, 24-well non-tissue culture plates werecoated with anti-CD3 and anti-CD28 antibodies diluted in PBS (1 μg/mL),sealed and incubated overnight at 4° C. Next day, rested PBMCs wereharvested, counted, washed and resuspended at the concentration of1×10⁶/ml. Antibody solution was aspirated, and wells were washed withcomplete media followed by addition of 2×10⁶ cells to each well.Activation was carried out at 37° C. for the specified time intervals.

Transduction

Activated T cells were harvested, washed and counted. Transductionmixtures containing concentrated virus supernatants, protamine sulfate(10 μg/ml), IL-7 (10 ng/ml) and IL-15 (100 ng/ml) were prepared. Foreach transduction, 1.0×10⁶ cells were separated in a sterilemicrocentrifuge tube and centrifuged at 400×g for 5 minutes. Each cellpellet was resuspended in 0.5 ml of the transduction mixturecorresponding to a specific MOI. Cell suspension was placed in anappropriately labelled well of a 24-well G-Rex plate. After 24 hours ofincubation at 37° C. and 5% CO₂, 1.5 ml media supplemented with IL-7 (10ng/ml) and IL-15 (100 ng/ml) was added to each well. Ninety-six-hourpost-transduction, transgene expression was determined by flowcytometry. Multimeric MHC-peptide complexes (Dextramer or Tetramer) wereused to monitor surface expression of transgenic TCR by FACS.

Flow Cytometry

Briefly, 1.0×10⁶ cells transduced at given lentiviral Multiplicity ofInfection (MOI) were stained following the work instructions. Fortetramer staining, cells were incubated with 1 μl of TAA tetramer in 50μl of Flow buffer for 15 minutes at RT in the dark. Tetramer stainingwas followed by staining with antibodies for T cells surface markers(e.g., CD3, CD4, CD8, etc). Samples were acquired with auto-compensationmatrix derived from compensation beads.

Results

PBMC obtained from 2 donors (D3 and D4) were activated for 16, 24 and 48hours using plate-bound anti-CD3 and CD28 antibodies. Cells weretransduced with 3 different lentiviral constructs (R72, R21, and R22).

FIG. 14A shows % CD3⁺CD8⁺Tetramer⁺ T cells gradually decrease with theincrease in the duration of activation in the order of 16h>24h>48h. Thisorder was consistently observed for both tested constructs and donors.

A time course study was performed to determine the optimal duration ofactivation for viral transduction that may result in high transgeneexpression. PBMC from one donor (D9) were activated with plate-boundanti-CD3 and anti-CD28 antibodies for the specified duration ofactivation, e.g., from 0 to 48 hours, and transduced with each of twodifferent lentiviral constructs encoding R7P1 D5 TCR, i.e., LV-R73 andLV-R78.

FIG. 14B shows transgene expression to be highest in cells activated for16-20 hours. Results represent level of transgene expression measured as% CD3⁺CD8⁺Tetramer⁺ T cells by flow cytometry 96-hour post transduction.The window for optimal activation was determined to be about 16 to about20 hours and may be extended to the maximum for 24 hours to addflexibility to GMP manufacturing.

Overall, these results may explain one of the causes of low transductionrates observed in T cells activated for 48 hours and show that shorteractivation for 16 to 24 hours may be optimal for performing lentiviraltransduction. Although, robust expansion achieved by 48-hour activationmay be impacted by limiting the activation time to 16 to 24 hours, thischange can be considered highly beneficial for the product andimplemented for all further process development and into the clinicalmanufacturing.

Example 3

Like all bioprocesses, scaling up of T cell manufacturing is achallenging part of the process development. Maintaining T cell functionand quality to preserve product efficacy is of prime importance throughall stages of scale up. For autologous T cell manufacturing process, thepresent inventors identified the critical steps and divided the scale-upinto two parts: scale up of activation may be carried out on anon-tissue culture surface and scale up of transduction and expansionmay be carried out in a G-Rex device.

Although beads or soluble antibodies present simpler methods to activatecells that are easily scalable, autologous T cell manufacturingprocesses which use immobilized antibodies on a non-tissue culturesurface (24-well plate) for activation yielded the best transduction andexpansion rates with the lentivirus. However, harvesting activated cellsfrom multiple 24-well non-tissue culture antibody coated plates posed tobe a laborious, time-consuming step that added complexity to anotherwise simple process. Considering activating up to 1×10⁹ PBMC, forexample, approximately 20 plates and 480 manipulations may be requiredto harvest activated cells in each manufacturing run.

Conventional methods of activating T cells may include an open-systemand a labor-intensive process using either commercially available beadsor non-tissue culture treated 24-well or 6-well plates coated withanti-CD3 and anti-CD28 antibodies (“plate-bound”) at a concentration of1 ug/mL each. Open system methods, however, may take a relatively longtime, e.g., about 8 hours, to complete. To simplify the open-system andthe labor-intensive process, embodiments of the present disclosure mayinclude a straightforward process adaptable to a closed-system that canbe combined with containers, e.g., bags, of commercially availableclosed system, e.g., G-Rex™ system and Xuri™ cell expansion system,resulting in comparable T cell activation profile, transducibility of Tcells, and functionality of the end-product with that of T cellsactivated using the conventional methods. In addition, methods of thepresent disclosure, e.g., flask-bound method, may take a relativelyshort time, e.g., about 1 hour, to complete, which is about 8 timesfaster than the conventional methods.

Optimizations for developing the autologous T cell manufacturing processwere performed at a small scale using 24-well non-tissue culture platesfor activation and 24-well G-Rex plates for transduction and expansion.At this scale, 1-2 million T cells transduced on Day 2 underwent from30-fold to 40-fold expansion until Day 10 to yield 30-80 million cellsat the time of harvest. However, the goal of the final process may be totransduce 250-400 million activated cells and expand them to over 10billion viable CD3⁺ T cells keeping the manufacturing timeline of 10days. Therefore, in an aspect, scaling up of entire process is providedherein.

For embodiments of the present disclosure including methods ofactivating larger number of cells, non-tissue culture treated T175cm²flasks provide a larger surface area and simpler platform requiring veryfew manipulations. After optimization, use of flask-bound antibodies foractivating T cells resulted in expansion and transduction comparable toplate-bound antibodies. Thus, scale up of the activation step was amajor development in simplifying the autologous T cell manufacturingprocess for clinical manufacturing. Based on greater cell numbers,transduction and expansion were scaled up from G-Rex-24 well plate toG-Rex 100. Use of G-Rex devices may facilitate nearly linear scale up ofpost-activation steps especially in terms of seeding density. Otherparameters, such as number of feeds and splits, may be standardized toachieve maximum expansion rates and viability. Validation of the entirescaled up process in full scale PD runs ensure successful technologytransfer of the T Cell Product #1 process in GMP.

Plate-Bound Versus Flask-Bound

T cell activation followed by transduction and expansion are criticalsteps of T cell manufacturing. To optimize the conditions for scaling upof these steps, use of T175cm² flasks presented a suitable platform foractivation with larger surface area and fewer manipulations to replace24-well plates coated with anti-CD3 and anti-CD28 antibodies. In acomparative study following optimization of critical parameters inT175cm² flasks, cells activated using antibodies coated on a flask(flask-bound) showed comparable levels of activation, transduction, andexpansion to cells activated using antibodies coated on 24-well plates(plate-bound) in multiple donors.

Further, transduction and expansion steps were scaled up from smallscale (G-Rex-24 well plate) to mid-scale (2-6 G-Rex10 or 1 G-Rex100) tofull scale (5-8 G-Rex100). The entire scaled-up process may be validatedin 2 full scale Process Development (PD) runs. All products generatedusing the final process passed the clinical release criteria in terms of% Dex⁺ CD3⁺CD8⁺ cells and generated cell numbers sufficient to meet theclinical doses.

Comparison Between T Cells Activated by the Plate-Bound Method and theFlask-Bound Method (a Non-Tissue Culture Treated Flask is Coated withAnti-CD3 and Anti-CD28 Antibodies) with Respect to Activation Level(Flow Cytometry), Transducibility (Dextramer Staining, FACS), Expansion(Cell Counts), and Functionality (IFN-γ ELISA)

PBMC from healthy donors were activated using anti-CD3 and anti-CD28antibodies using non-tissue culture treated T175cm² flasks or 24-wellplates in preparation for transduction. Activated T cells weretransduced with a lentiviral construct encoding R7P1 D5 TCR and seededin G-Rex 24-well plates or G-Rex10/G-Rex100 flasks. Transduced T cellswere expanded in the presence of IL-7 and IL-15 and harvested on Day 10of the process. In-process and final testing were performed on theproducts to determine cell counts, viability and percentage oftransduced CD8⁺ T cells.

Representative Materials and Methods

Supplies Manufacturer Catalog # TexMACS media Miltenyi Biotec130-097-196 Human AB Serum Gemini 100-512 IL-7 Peprotech 200-07 IL-15Peprotech 200-15 Anti-CD3 antibody Ebioscience 16-0037-85 Anti-CD28antibody Ebioscience 16-0289-85 24-well non-tissue culture platesCo-star 3738 T175 cm² non-tissue culture plates Corning 431466 G-Rex24-well plate Wilson Wolf 80192M G-Rex10 Wilson Wolf 80040S G-Rex100Wilson Wolf 80500S 15 mL Conical Tube Falcon 352097 50 ml conical tubeCorning 430290 5 mL serological Pipet BD 53300-421 10 mL serologicalPipet BD 53300-523 25 ml serological pipet BD 53300-567 1000 ul pipettips Rainin 17007954 200 ul pipet tips Rainin 17007961 20 μL pipet tipsRainin 17007957 1.5 mL Microcentrifuge Tube Fisher 02-681-5 AOPIStaining Solution Nexcelom CS2-0106 PBS without Mg and Ca Lonza17-516F/24 96 well plate Corning 3799 P-20 Micropipettor Rainin 17014382P-200 Micropipettor Rainin 17014391 P-1000 Micropipettor Rainin 17017382Pipettaid Drummond 193970L T75 flasks BD Falcon BD353136 Benzonase SigmaE1014 Protamine sulfate McKesson 804514 Lentivirus Lentigen LV-R73, R78Live/Dead Aqua dye Thermo Fisher L-34966 ABC Comp Beads Thermo FisherA10497 CD3-BV421 BD 562426 CD8-APC Biolegend 301014 CD4-PerCPCy5.5 BD560650 TAA Dextramer-PE Immudex N/A

Methods

Experiments were carried out following the standard autologous T cellmanufacturing process involving 4 steps: thaw/rest, activation,transduction, and expansion, however at different scales.

Thaw and Rest

Frozen PBMC from healthy donors were thawed in warm TexMACS mediasupplemented with 5% human AB serum (complete media). Cells were treatedwith benzonase nuclease (50U/ml) for 15 minutes at 37° C., washed,counted and put to overnight rest in complete TexMACS media.

Activation

On the day of thawing cells, 24-well non-tissue culture plates orT175cm² flasks were coated with anti-CD3 and anti-CD28 antibodiesdiluted in PBS (1 μg/mL), sealed and incubated overnight at 4° C. Nextday, rested PBMCs were harvested, counted, washed and resuspended at theconcentration of 1×10⁶/ml. Antibody solution was aspirated, and wellswere washed with complete media followed by addition of 2×10⁶ cells toeach well. Activation was carried out at 37° C. for the specified timeintervals.

Transduction

Activated T cells were harvested, washed and counted. Transductionmixtures containing preclinical lentiviral supernatants (calculatedbased on a specified MOI), protamine sulfate (10 μg/ml) and IL-7 (10ng/mL) and IL-15 (100 ng/mL). For each transduction, activated cellswere separated and centrifuged at 400×g for 5 minutes. Each cell pelletwas resuspended in the transduction mixture (1 ml per 2×10⁶ cells) andseeded in an appropriately sized G-Rex flask. After 24 hours ofincubation at 37° C. and 5% CO₂, culture volume in each G-Rex flask wasbrought to half or full capacity as specified using media supplementedwith IL-7 (10 ng/ml) and IL-15 (100 ng/ml). Cell counts and viabilitywere monitored regularly up to Day 10 of the process. MultimericMHC-peptide complexes (Dextramer or Tetramer) were used to monitorsurface expression of transgenic TCR by FACS.

Flow Cytometry

Briefly, 1.0×10⁶ transduced cells were stained following the workinstructions. Tetramer staining was followed by staining with antibodiesfor T cells surface markers. Samples were acquired withauto-compensation matrix derived from compensation beads.

Results

To evaluate non-tissue culture treated T175cm² flasks as an alternativeto 24-well plates for coating anti-CD3 and anti-CD28 antibodies toactivate T cells, the antibody concentration was kept the same as insmall scale, other parameters such as coating volume, cell density, andseeding volume were optimized for a larger area in a flask.

To compare viability and expression of activation markers CD25 and CD69and LDL-R in plate-bound (PB) and flask-bound (FB) activated PBMC, FACSstaining and acquisition were performed 16-24 hours post-activationusing FB or PB anti-CD3 and CD28 antibodies. Unstimulated PBMC were usedas negative controls.

FIG. 15 shows, under optimized conditions, T cells activated in T175cm²flasks (flask-bound, FB) exhibit comparable expression levels ofactivation markers CD25 and CD69 and LDL-R to that of T cells activatedunder plate-bound (PB) conditions. These results suggest scale-upactivation using FB antibodies may be feasible in view of the comparablelevels of activation resulting from FB and PB activated T cells.

To compare transgene expression and expansion in Day 10 harvested T cellproducts (from donor 6, donor 7, and donor 8) using PB or FB antibodiesfor activation, surface expression of R7P1 D5 TCR was determined by flowcytometry using TAA specific dextramer or transgenic TCR 13 chainspecific antibody. Fold expansion was calculated on the basis of viablecell number seeded in the G-Rex plate or flask at the time oftransduction (Day 2) and the day of harvest (Day 10). FIGS. 16A and 16Bshow comparable levels of transduction and fold expansion, respectively,in FB and PB activated T cells. These results suggest scale-uptransduction and expansion using FB antibodies may be feasible in viewof the comparable levels of transduction and expansion resulting from FBand PB activated T cells.

For further validation of successful scale up of activation,functionality of T cell products generated by FB and PB activationmethods were compared. To evaluate induction of antigen specific IFN-γby LV-R73 transduced T cell products generated using PB or FB antibodiesfor activation, IFN-γ released in the supernatant of T cell co-culturedwith tumor cell lines (Target+ve, Target−ve) was quantitated using acommercially available ELISA kit.

FIG. 17 shows FB activated LV-R73 transduced T cells secreted comparablelevels of antigen specific IFN-γ to that of PB activated transduced Tcells in response to tumor cells expressing TAA in each donor 6, donor,7, and donor 8. These results suggest scale-up of IFN-γ-secreting Tcells using FB antibodies may be feasible in view of the comparablelevels of IFN-γ secretion resulting from FB and PB activated T cells.

For scale up of the remaining process, 2.5×10⁸-4.0×10⁸ activated T cellswere transduced and seeded at optimal seeding density of 0.5×10⁶ per cm²of surface area of the G-Rex100 flask. Multiple G-Rex100 flasks wereused to seed the transduced cells at the optimal density. Additionalparameters, such as conditions for feeding and splitting the cells, werealso optimized to achieve maximum expansion. The final manufacturingprocess was tested in 2 full scale Process Development (PD) runs. Allproducts generated using the final process passed the % Dextramer andintegration copy number release criteria. Cell numbers generated inthese manufacturing runs met clinical dose at all cohort levels. Resultsof PD scale up runs are summarized in the Table 1 below.

TABLE 1 Summary of product characterization from 2 full scale PD runsperformed Scale up % % % Integration Run# Donor CD3 CD8 Dextramer⁺ Copy#Cell # 1 Donor 6 99.5% 66.9% 21.6% 0.96 1.01 × 10¹⁰ 2 Donor 9 96.2%57.7% 23.1% 1.21 1.05 × 10¹⁰

GMP manufacturing with the above process have yielded over 20 billioncells for a few donors.

Flask-Bound Versus Bag-Bound

Comparison Between T Cells Activated by Flask-Bound Method and Bag-BoundMethod (e.g., Saint-Gobain VueLife AC Bag Coated with Anti-CD3 andAnti-CD28 Antibodies) with Respect to Activation Level (Flowcytometry),Transducibility (Dextramer Staining, FACS), and Expansion (Cell Counts)

To compare activation of T cells using anti-CD3 and anti-CD28 antibodycoated bags versus plates, FIG. 18 shows the experimental design used totest the effect of anti-CD3 and anti-CD28 antibody coated bags andplates on T cell activation. Briefly, on Day 0, PBMC were thawed andrested overnight (24 hours). On Day 1, the rested PBMC were activated byseeding them on flasks, e.g., T175cm² flasks, or bags, e.g.,Saint-Gobain VueLife AC Bags, coated with anti-CD3 and anti-CD28antibodies for 16-20 hours. On Day 2, activated T cells were analyzedfor CD25, CD69, and hLDL-R expression and transduced with VSV-Gpseudotyped lentiviral vectors, e.g., 1×Eng LV-R73. On Day 6/7 and 10,analyses, such as cell counts, viability, and fluorescence-activatedcell sorting (FACS) with dextramers (Dex), were performed.

FIG. 19 shows, under optimized conditions, T cells (from donor 16 anddonor 14) activated in bags bound with antibodies at concentrations of 1μg/ml or 2 μg/ml exhibit comparable expression of activation markersCD25 and CD69 and hLDL-R expression to those activated under flask-bound(T175cm² flask, labelled as “standard”) conditions. These resultssuggest scale-up activation using bag-bound (Bag) antibodies may befeasible in view of the comparable levels of activation resulted frombag-bound (Bag) and flask-bound (FB) activated T cells.

FIGS. 20 and 21 shows, on Day 6 and Day 10 of expansion, respectively, Tcells (from donor 16 and donor 14) activated in bags bound withantibodies at concentrations of 1 μg/ml or 2 μg/ml exhibit comparable Tcell counts, i.e., cell expansion, with that of T cells activated underFB conditions. These results suggest scale-up expansion using bag-boundantibodies may be feasible in view of the comparable levels of expansionresulting from bag-bound and flask-bound activated T cells.

Example 4

Short Rest Versus Overnight Rest in T Cell Manufacturing Process

FIG. 22 shows a T cell manufacturing process 220 by resting PBMC for aperiod of time of about 4 hours according to one embodiment of thepresent disclosure. For example, a T cell manufacturing process 220 mayinclude Isolation and cryopreservation of PBMC from leukapheresis (221),in which sterility may be tested; thaw, rest (e.g., about 4 hours) andactivate T cells (222); transduction with a viral vector (223);expansion with cytokines (224); split/feed cells (225), in which cellcount and immunophenotyping may be tested; harvest and cryopreservationof drug product cells (226), in which cell count and mycoplasma may betested, and post-cryopreservation release (227), in which viability,sterility, endotoxin, immunophenotyping, copy number of integratedvector, and vesicular stomatitis virus glycoprotein G (VSV-g) may betested.

Table 2 shows characteristics of T cells manufactured by three differentqualification runs of T cell manufacturing process 220 by resting PBMCfor a short period of time, e.g., about 4 to about 6 hours, in thepresence of IL-7 according to one embodiment of the present disclosure.

TABLE 2 Qualification runs (QR) of T cell manufacturing process 220 byresting PBMC for a short period of time, e.g., about 4 to about 6 hours,preferably about 4 hours (in GMP cleanroom) QR1 QR2 QR3 Average % CD3+99.6 99.7 99.8 99.7 % CD8+ 33.5 51.5 75.2 53.4 % Dex+/CD3+CD8+ 35.5 72.783.0 63.7 % Viability 92.0 92.2 91.7 92.0 Residual VSV-g <50 <50 <50 <50copies/μg copies/μg copies/μg copies/μg Average copy  1.0  3.0  4.2  2.7number (per cell) Total viable cells 24.8 × 10⁹ 32.2 × 10⁹ 26.8 × 10⁹28.0 × 10⁹ Transduced cells 2.95 × 10⁹ 12.1 × 10⁹ 16.73 × 10⁹ 10.6 × 10⁹Days manufacturing 10  8  8  8.7 Cells at transduction 281 × 10⁶ 400 ×10⁶ 400 × 10⁶ 360 × 10⁶ (max) (max) Fold expansion 88-fold 81-fold67-fold 78.7-fold LV batch ENG ENG GMP NA

FIG. 23A and Table 2 show average fold expansion of T cells (n=7)manufactured by resting PBMC overnight is about 78.7-fold.

To determine whether transduced TCR is expressed on the cell surface ofexpanded T cells, expanded T cells were stained with peptide/MHCcomplex-loaded dextramers that specifically bind to transduced TCR,followed by flow cytometry to identify CD8+ T cells expressingtransduced TCR. FIG. 23B and Table 2 show average % Dex+/CD8⁺ T cells(n=7) manufactured by short rest is about 53.4%, indicating thattransduced TCR is expressed on the cell surface of expanded T cells.

To determine what T cell phenotypes are present in expanded T cellsexpressing transduced TCR, cells were stained with various immune cellsurface markers, followed by flow cytometry to identify T cellphenotypes, e.g., T_(n/scm), T_(cm), T_(em), and T_(eff). Among them,T_(n/scm) may be more desirable for immunotherapy than others becauseT_(n/scm) may have properties of lymphoid homing, proliferationpotential, self-renewal, and multipotency. FIG. 23C shows average about50% of expanded T cells expressing transduced TCR (n=4) exhibitingT_(n/scm) phenotypes.

To determine cytotoxic activity of expanded T cells expressingtransduced TCR, tumor cells pulsed with different concentration oftarget peptide were incubated with expanded T cells expressingtransduced TCR that specifically recognizes target peptide/MHC complex,followed by measuring tumor cell growth. FIG. 23D shows expanded T cellsexpressing transduced TCR inhibit tumor cell growth in a peptideconcentration dependent manner.

Cytotoxic activities of expanded T cells expressing transduced TCRappear comparable between PBMC obtained from different healthy donors,e.g., Donors 7, 13, 17, 18, and 21 (FIG. 23E), and that obtained fromdifferent patients, e.g., Patients 312, 319, 351, 472, and 956 (FIG.23F).

To determine cytotoxic potential of the expanded T cells expressingtransduced TCR, tumor cells expressing target peptide were incubatedwith expanded T cells expressing transduced TCR (220-T) thatspecifically recognize target peptide/MHC complex, followed by measuringfold growth of tumor cells. FIG. 23G and FIG. 23H show increasedregression or suppression of tumor growth by incubation with expanded Tcells expressing transduced TCR (220-T) (effectors) at effectors totumor cells ratios of 10:1 and 3:1 as compared with that of thenon-transduced T cells lacking target-specific TCR (NT).

FIG. 24 shows a T cell manufacturing process 240 by resting PBMCovernight (about 16 hours). For example, T cell manufacturing process240 may include isolation of PBMC (241), in which PBMC may be used freshor stored frozen till ready for use, or may be used as startingmaterials for T cell manufacturing and selection of lymphocytepopulations (e.g., CD8, CD4, or both) may also be possible; thaw andrest lymphocytes overnight, e.g., about 16 hours, (242), which may allowapoptotic cells to die off and restore T cell functionality (this stepmay not be necessary, if fresh materials are used); activation oflymphocytes (243), which may use anti-CD3 and anti-CD28 antibodies(soluble or surface bound, e.g., magnetic or biodegradable beads);transduction with CAR or TCR (244), which may use lentiviral orretroviral constructs encoding CAR or TCR or may use non-viral methods;and expansion of lymphocytes, harvest, and cryopreservation (245), whichmay be carried out in the presence of cytokine(s), serum (ABS or FBS),and/or cryopreservation media.

Table 3 shows characteristics of T cells manufactured by three differentqualification runs of a T cell manufacturing process (240) by restingPBMC overnight, e.g., about 16 hours.

TABLE 3 Qualification runs (QR) of a T cell manufacturing process byresting PBMC overnight (in GMP cleanroom) QR1 QR2 QR3 Average % CD3+99.2 99.6 99.7 99.5 % CD8+ 47.9 46.9 60.9 51.9 % Dex+/CD3+CD8+ 36.7 57.364.9 53.0 % Viability 85.6 86.8 85.5 86.0 Residual VSV-g <50 <50 <50 <50copies/μg copies/μg copies/μg copies/μg Average copy 2.7 3.2 3.6 3.2number (per cell) Total viable cells 8.7 × 10⁹ 24.3 × 10⁹ 14.2 × 10⁹15.7 × 10⁹ Transduced cells 1.3 × 10⁹ 5.65 × 10⁹ 4.8 × 10⁹ 3.9 × 10⁹Days manufacturing 8 9 8 8.3 Cells at transduction 231 × 10⁶ 400 × 10⁶400 × 10⁶ 344 × 10⁶ Fold expansion 38-fold 61-fold 36-fold 45-fold LVbatch ENG ENG GMP NA

In contrast to T cell manufacturing process with short rest, e.g., about4 hours, T cells manufactured with rest of about 16 hours yielded lessfold expansion of T cells. FIG. 25A and Table 3 show average foldexpansion of T cells (n=7) manufactured by resting PBMC for about 16hours is about 45-fold, as compared with about 78.7-fold with short restof about 4 hours (Table 2). TT and PQ stand for Technology Transfer andProcess Qualification runs, respectively.

Overnight rest (about 16 hours) yielded less expanded T cells expressingtransduced TCR than rest of about 4 hours. FIG. 25B and Table 3 showaverage % Dex+/CD8+ T cells (n=7) manufactured by resting PBMC overnightfor about 16 hours is about 51.9%, as compared with about 53.4% withrest of about 4 hours (Table 2).

Overnight rest of about 16 hours yielded less expanded T cellsexpressing transduced TCR with T_(n/scm) phenotype than rest of about 4hours. FIG. 25C shows average about 40% of expanded T cells (n=5) havingT_(n/scm) phenotypes, as compared with about 50% with rest of about 4hours (FIG. 23C).

FIG. 25D shows significantly more inhibition of tumor cell growth byincubation of tumor cells with expanded T cells expressing transducedTCR (effectors) at effectors to tumor cells ratios of 10:1, 3:1, and 1:1than that of the negative controls, e.g., tumor cells incubated witheither expanded T cells that do not express transduced TCR (Target-ve)or no effectors. In addition, cytotoxic activities of expanded T cellsexpressing transduced TCR appear comparable between PBMC obtained fromhealthy donors (n=5) (FIG. 25E) and that obtained from cancer patients(n=7) (FIG. 25F).

Table 4 summarizes characteristics of T cells manufactured with shortrest of about 4 hours according to one embodiment of the presentdisclosure (process 220) and that with overnight rest of about 16 hours(process 240).

TABLE 4 % % Dex+ % Live CD8+ of Fold Harvest Viability ≥ CD3+ ≥ of CD8+≥ Process Expansion Count 70% 80% CD3+ 10% 220 78.7 28.0 × 10⁹ 92.0 99.753.4 63.7 240 45.0 15.7 × 10⁹ 86.0 99.5 51.9 53.0

Table 4 shows process with short rest 220 (about 4-6 hours) may allow anextra day in expansion, e.g., Day 8 of process 240 is Day 9 for process220, thus, resulting in more cells.

Example 5

T Cell Manufacturing in Closed System

As noted above, processes 220 and 240 may be carried out in opensystems, such as G-Rex™. Ex vivo manipulation of haematopoietic cells,e.g., T cells, in open systems, however, may introduce risk ofcontamination with infectious agents and may reduce engraftmentpotential and haematopoietic fitness. In manufacturing clinical cellproducts, closed cell culture systems may be preferred due to theassurance of sterility throughout culture processes.

FIG. 26 shows ex vivo manipulation protocol in open and closed systems.Closed systems not only can mitigate external processing risks andcontamination, but also promote product robustness and quality, andincrease product security, thus, can reduce challenges for downstreamprocessing, final product analysis, and testing. While relatively smallnumbers of cells, e.g., ≤1×10⁹, may be cultured in a relatively smallvolume in open system, e.g., 1 liter, relatively large numbers of cells,e.g., from about 1×10⁹ to about 2×10¹¹, may be cultured in a relativelylarge volume in closed system, e.g., from 5 liters (e.g., WAVE (XURI™)Bioreactor bag and G-Rex™ flask) to 50 liters (e.g., static bag). Theseclosed system cell culturing technologies may deliver high quality,individualized cell therapies as a regulated, faster, and cost-effectiveroute of cell manufacturing.

T cell manufacturing process of the present disclosure may be carriedout in any cell culture closed systems including commercially availablesystems, e.g., CliniMACS Prodigy™ (Miltenyi), WAVE (XURI™) Bioreactor(GE Biosciences) alone or in combination with BioSafe Sepax™ II, andG-Rex/GatheRex™ closed system (Wilson Wolf) alone or in combination withBioSafe Sepax™ II. G-Rex™-closed system is the expansion vessel andGatheRex™ is the pump for concentrating and harvesting.

CliniMACS Prodigy™ (Miltenyi)

CliniMACS Prodigy™ with TCT process software and the TS520 tubing setmay allow closed-system processing for cell enrichment, transduction,washing and expansion. For example, MACS-CD4 and CD8-MicroBeads may beused for enrichment, TransACT beads, e.g., CD3/CD28 reagents, may beused for activation, lentiviral vectors expressing a recombinant TCR maybe used for transduction, TexMACS medium-3%-HS-IL2 for culture andphosphate-buffered saline/ethylenediaminetetraacetic acid buffer forwashing. This system may yield about 4-5×10⁹ cells, contain automatedprotocols for manufacturing with chamber maximum ˜300 mL fill volume,and perform selection and activation (TransACT beads), transduction, andexpansion over a 10 to 14-day process.

WAVE (Xuri™) Bioreactor (GE Biosciences)

WAVE (Xuri™) Bioreactor allows T cells to be cultured in culture bags,e.g., Xuri Cellbags, with and/or without perfusion. Medium bag forfeeding may be 5-liter Hyclone Labtainer. Waste bag may be Mbag(purchased from GE Healthcare). This system may yield about 15-30×10⁹cells, use unicorn software that allows for culture control andmonitoring, contain rocking tray that may hold from about 0.3-liter toabout 25 liters, and perform perfusion function to maintain culturevolume while mediating gas exchange and introducing fresh media andcytokines to cell culture.

WAVE (Xuri™) Bioreactor may include Xuri Bags for expansion, SaintGobain's VueLife bags for thawing and resting, and VueLife AC bags foractivation. WAVE (Xuri™) Bioreactor may be used in combination withother technologies, e.g., Sepax™ cell separation system (GE Biosciences)for culture washing and volume reduction steps. Sterile welder (TerumoBCT™) may be used for connecting sterile bags for solution transfer andheat sealer for sealing tubing.

Sepax™ cell separation system relies on a separation chamber thatprovides both separation through rotation of the syringe chamber(centrifugation) and component transfer through displacement of thesyringe piston. An optical sensor measures the light absorbency of theseparated components and manages the flow direction of each of them inthe correct output container, for example, plasma, buffy coat, and redblood cells may be thus separated and collected from blood samples.

FIG. 27 shows, on Day 0, frozen PBMC isolated by Sepax™ cell separationsystem may be thawed, washed, rested, e.g., overnight (O/N), and culturebags, e.g., VueLife AC cell bags, may be coated with anti-CD3 antibodyand anti-CD28 antibody; on Day 1, rested PBMC may be transferred toculture bags coated with anti-CD3 antibody and anti-CD28 antibody foractivation; on Day 2, cells may be washed and media may be reduced bySepax™ cell separation system to an appropriate volume suitable forviral transduction, e.g., transduced with lentiviral vector expressingTCR. Cell expansion can be performed in Xuri™ culture bags on a rockingtray with perfusion function to maintain culture volume while mediatinggas exchange and introducing fresh media and cytokines to cell culture.Expanded transduced T cells may be harvested and washed using Sepax™cell separation system.

G-Rex/GatheRex™ Closed System (Wilson Wolf)

G-Rex/GatheRex™ closed system comprises a gas-exchange vessel (G-Rex-CS)for cell expansion and an automated pump (GatheRex) that may allow theoperator to drain the excess media present in the culture and collectcells without risk of contamination. The harvesting process may bedivided into two stages: cell concentrating and cell harvesting. In cellconcentrating process, GatheRex™ closed system may operate via an airpump, which pressurizes the G-Rex™ device, e.g., flasks, with sterileair, allowing 90% of the medium residing above the cells to be displacedinto a medium collection bag. Once this process is complete, a firstoptical detector senses the presence of air in the medium collectionline, automatically stopping the pump. Prior to beginning the harvestprocess, the operator may resuspend the cells using the residual 10% ofthe medium by manually swirling the G-Rex™ device to dislodge cells fromthe gas-permeable membrane. The air pump is then reactivated, and theresuspended cells are drawn into the cell collection bag. This phase mayautomatically end once a second optical detector detects air in the cellcollection line. This system may yield about 15-20×10⁹ cells and hold5-liters per vessel.

G-Rex/GatheRex™ closed system may support transduction and expansion inthe vessel and harvest with the pump. Thawing, resting, and activationsteps may be carried out in VueLife™ bags. GatheRex™ closed system maybe used in combination with other technologies, e.g., Sepax™ cellseparation system for culture washing and volume reduction stepsSterilewelder (Terumo BCT™) may be used for connecting sterile bag for solutiontransfer and heat sealer for sealing tubing.

FIG. 28 shows on Day 0, frozen PBMC isolated by Sepax™ cell separationsystem may be thawed, washed, rested, e.g., overnight (O/N); on Day 1,culture bags may be coated with anti-CD3 antibody and anti-CD28 antibodyand rested PBMC may be transferred to the coated culture bags foractivation; on Day 2, cells may be washed and media may be reduced bySepax™ cell separation system to an appropriate volume suitable forviral transduction, e.g., transduced with lentiviral vector expressingTCR. Cell expansion and feeding may be performed in G-Rex™ closed systemdevices. Expanded transduced T cells may then be harvested using theGatheRex™ pump and washed using Sepax™ cell separation system.

Table 5 shows comparison between T cells obtained by open systems, e.g.,G-Rex™, as shown in Table 4, i.e., processes 220 and 240, and T cellsobtained by closed systems, e.g., CliniMACS Prodigy™, WAVE (XURI™)Bioreactor in combination with BioSafe Sepax™ II, and G-Rex/GatheRex™closed system in combination with BioSafe Sepax™ II.

TABLE 5 % Live % Dex+ of Fold Viability ≥ CD3+ ≥ % CD8+ CD8+ ≥ ProcessExpansion Harvest Count 70% 80% of CD3+ 10% 220 78.7 28.0 × 10⁹ 92.099.7 53.4 63.7 240 45.0 15.7 × 10⁹ 86.0 99.5 51.9 53.0 CliniMACS 55.0 4.4 × 10⁹ 95.4 98.5 55.0 39.7 Prodigy ™ WAVE 40.3 16.1 × 10⁹ 92.0 99.660.8 41.7 (XURI ™) Bioreactor in combination with BioSafe Sepax ™ IIG-Rex/ 46.3 18.5 × 10⁹ 89.7 99.4 62.8 49.5 GatheRex ™ in combinationwith BioSafe Sepax ™ II

These results show T cell manufacturing process of the presentdisclosure can be readily performed in closed systems to produce T cellswith comparable characteristics to that produced in open systems, whilemitigating external processing risks and contamination, promotingproduct robustness and quality, and increasing product security, andthus, reducing challenges for downstream processing, final productanalysis, and testing.

To further compare functional characteristics of engineered T cellsmanufactured in closed systems with that manufactured in open systems,PBMCs obtained from donor 17 were processed to produce expandedtransduced T cells according to the process of the present disclosure.The expanded transduced T cells expressing TCR were then measured forIFN-γ release in the present or absence of TCR-specific peptide/MHCcomplex (target).

FIG. 29 shows that engineered T cells manufactured in closed systems asmeasured by two runs, Run #1 and Run #2, released significantly moreIFN-γ in the presence of target than that manufactured in open system,e.g., process 220. These results suggest that engineered T cellsmanufactured in closed systems may exhibit greater cytotoxic activitythan that manufactured in open systems.

Example 6

GMP Manufacturing of TCR-Engineered T Cells in about 5 to 6 Days

Adoptive cellular therapy with autologous engineered T cells approachcapitalizes on translational development of safe and effective targetsand their cognate TCRs. These TCRs are genetically engineered intopatients' own (autologous) T-cells for the immunotherapy of solidtumors.

FIG. 30 shows manufacturing outline of three T-cell products (T CellProduct #1, T Cell Product #2, and T Cell Product #3) each expressing atransgenic TCR against its own respective HLA-A*02:01 restricted tumortargeted antigen. T Cell Product #1 and T Cell Product #2 weremanufactured in about 8-11 days and about 7-10 days, respectively, fromthawing frozen PBMC, resting the thawed PBMC, and activating the restedPBMC (Step 2), transducing the activated T cells (Step 3), to “harvestand cryopreservation of drug product cells” (Step 6), using open systemsfor IND driven phase 1 first in man trials.

T Cell Product #3 may be manufactured by shortening the expansion phasefrom about 5-8 days (T Cell Products #1 and #2) to about 3-4 days. Inaddition, T Cell Product #3 may be manufactured by activating freshPBMC, i.e., PBMC is not cryopreserved and then thawed, on Day 0. This isin contrast to the manufacturing T Cell Products #1 by thawing thecryopreserved PBMC on Day 0 and then activating the thawed PBMC on Day 1and the manufacturing T Cell Products #2 by thawing the cryopreservedPBMC and activating the thawed PBMC on Day 0.

In contrast to T Cell Products #1 and #2, which are manufactured byusing open systems, T Cell Product #3 may be manufactured by using acomplete closed system or a semi-closed system, in which some steps maybe performed by using open systems, e.g., from T cell activation tovolume reduction for transduction and/or from harvest to washing,concentration, and cryopreservation.

FIG. 31 shows the turnaround time from leukapheresis collection toinfusion-ready TCR T Cell Product #1 may take about 30 days, e.g., about14 days from sample collection to harvest and about 16 days from qualitycontrol (QC) to product release; and the turnaround time formanufacturing TCR T Cell Product #2 may take about 26 days, e.g., about10 days from sample collection to harvest and about 16 days from QC toproduct release.

There is, however, a need for fast turnaround. FIG. 30 shows, T CellProduct #3 was manufactured using shorter manufacturing process, e.g.,5-6 days, from “optional thaw, rest, and activation” (Step 2) to“harvest and cryopreservation of drug product cells” (Step 6), usingsemi-closed system. FIG. 31 shows TCR T Cell Product #3 may take about23 days to manufacture, e.g., about 7 days from sample collection toharvest and about 16 days from QC to product release. For commercialmanufacturing, for example, TCR T cell products, e.g., T Cell Product#1, T Cell Product #2, and T Cell Product #3, may take about 13 days tomanufacture, e.g., about 6 days from sample collection to harvest andabout 7 days from QC to product release.

FIG. 32 shows a T cell manufacturing process 320 using fresh PBMCs,which is not obtained by thawing cryopreserved PBMC, thus, minimizingcell loss due to freezing, thawing, and/or resting PBMCs and maximizingcell numbers at the beginning of manufacturing process. For example, Tcell manufacturing process 320 may include Day 0, isolation of freshPBMC (321), activation of fresh lymphocytes (322) using, for example,anti-CD3 and anti-CD28 antibodies (soluble or surface bound, e.g.,magnetic or biodegradable beads) in bags, e.g., Saint-Gobain VueLife ACBags, coated with anti-CD3 and anti-CD28 antibodies; Day 1, transductionwith CAR or TCR (323) using, for example, lentiviral or retroviralconstructs encoding CAR or TCR or non-viral methods, e.g., liposomes;and Day 2, expansion of lymphocytes, Day 5/6, harvest, andcryopreservation (324) in the presence of cytokine(s), serum (ABS orFBS), and/or cryopreservation media.

Improved Product Profile with Shorter Expansion

The quality, efficacy, longevity, and location of T cell immunity mayresult from the diversification of naive T cells (T_(n)) into variousphenotypically distinct subsets with specific roles in protectiveimmunity. These include memory stem (T_(scm)), central memory (T_(cm)),effector memory (T_(em)), and highly differentiated effector (T_(eff)) Tcells. The antigen-specific T_(n) give rise to long-lived T_(scm) andTail that self-renew and provide proliferating populations ofshorter-lived T_(em) and T_(eff) cells. Therefore, selecting lessdifferentiated T_(n), T_(scm) or T_(cm) subsets for genetic modificationmay provide cells with greater therapeutic efficacy.

To evaluate the differentiation status of T cell products harvested atdifferent time of manufacturing, CD8+ T cells obtained from 3 donors(Donor 1, Donor 2, and Donor 3) were harvested on Day 4 (expansion for 3days), 7 (expansion for 6 days) and 10 (expansion for 9 days) ofmanufacturing followed by T cell memory phenotyping analysis.

FIG. 33 shows the amount of CD8+ T cells exhibiting the lessdifferentiated phenotypes, e.g., T_(n/scm)−CD45RA+CCR7+ andT_(cm)−CD45RO+CCR7+, decreases in an expansion time-dependent manner,i.e., Day 4>Day 7>Day 10. Conversely, the amount of CD8+ T cellsexhibiting the more differentiated phenotypes, e.g., T_(em)−CD45RO+CCR7−and T_(eff)−CD45RA+CCR7−, increases in an expansion time-dependentmanner, i.e., Day 4<Day 7<Day 10, indicating more less differentiatedphenotypes of Day 4 expanded cells than that of Day 7 and Day 10expanded cells. These results suggest the shorter the T cells expand,the more the T cells exhibit less differentiated memory phenotypes,thus, with greater therapeutic efficacy.

CD27 and CD28 co-stimulation may be required during primary CD8+ T cellresponses. This co-stimulation may provide proliferation and survivalcues to naive CD8+ T cells. To evaluate the CD27 and CD28 co-stimulationpotentials of T cell products harvested at different time ofmanufacturing, CD8+ T cells obtained from 3 donors (Donor 1, Donor 2,and Donor 3) were harvested on Day 4, 7 and 10 of manufacturing followedby CD27 and CD28 expression analysis.

FIG. 34 shows the amount of CD8+ T cells exhibiting the CD27+CD28+co-stimulation phenotypes decreases in an expansion time-dependentmanner, i.e., Day 4>Day 7>Day 10, indicating superior CD27 and CD28co-stimulation of Day 4 expanded cells to that of Day 7 and Day 10expanded cells. These results suggest, in general, the shorter the Tcells expand, the more the T cells express both CD27 and CD28.

To evaluate the replicative potentials of T cell products harvested atdifferent time of manufacturing, T cells were harvested on Day 4, 7 and10 of manufacturing and monitored for growth in response to relevantcytokines, e.g., IL-7, IL-15, or IL-2 in cytokine sensitivity assay.

FIG. 35 shows T cell growth induced by IL-7, IL-15, or IL-2 for about 21days decreases in an expansion time-dependent manner, i.e., Day 4>Day7>Day 10, indicating superior replicative potentials of Day 4 expandedcells to that of Day 7 and Day 10 expanded cells. These results suggestthe shorter the T cells expand, the more the T cells respond tocytokines for proliferation.

To evaluate the anti-tumor activity of T cell products harvested atdifferent time of manufacturing, T cell products obtained from 4 donors(Donor 1, Donor 2, Donor 3, and Donor 4) were harvested on Day 5, 7 and9 of manufacturing followed by interferon-gamma (IFN-γ) release assaysin response to exposure to target positive cell line.

FIG. 36 shows IFN-γ secretion decreases in an expansion time-dependentmanner, i.e., Day 5>Day 7>Day 9, indicating, in general, superioranti-tumor activity of Day 5 expanded cells to that of Day 7 and Day 9expanded cells. These results suggest, in general, the shorter the Tcells expand, the more the T cells secret IFN-γ.

To further evaluate the cytotoxic activity of T cell products harvestedat different time of manufacturing, EC₅₀ based on IFN-γ response againstT2 cells pulsed with decreasing concentrations of the cognate peptidewas determined.

FIG. 37 shows EC₅₀ increases in an expansion time-dependent manner,i.e., Day 5>Day 7>Day 9, indicating superior peptide-specific cytotoxicactivity of Day 5 expanded cells to that of Day 7 and Day 9 expandedcells.

T Cell Product #3 GMP Manufacturing

Characterization of Products Manufactured with Final T Cell Product #3Process

FIG. 38 shows expansion metrics. In two Technology Transfer (TT)manufacturing runs and two Process Qualification (PQ) manufacturing runs(n=4), an average of 1.3×10¹⁰ cells was harvested with >90% viabilityfollowing short expansion, e.g., about 6 days.

FIG. 39 shows surface expression of T Cell Product #3 TCR detected byflow cytometry using a TCR specific HLA-dextramer. A representative FACSplot and combined data (Mean±SD) are shown from Technology Transfer (TT)and Process Qualification (PQ) manufacturing runs (n=4) performed usingleukapheresis products from healthy donors.

FIG. 40 shows T-cell memory phenotype of the final T Cell Product #3, inwhich T cells produced by Technology Transfer (TT1, TT2) and ProcessQualification (PQ1, PQ2) manufacturing runs preserve less differentiatedphenotype in donors representing highly variable memory phenotype of Tcell populations in PBMC used for manufacturing (n=4) (T_(n/scm)—17.9%,19.2%, 11.2%, 35.0% T_(cm)—23.4%, 15.7%, 0.9%, 2.4% T_(em)—34.8%, 27.0%,25.9%, 43% T_(eff)—23.8%, 38.2%, 62.0%, 16.1% respectively)

FIG. 41 shows IFN-γ release in response to exposure to target positive(LVR11KEA) and negative (NT) cell lines. T cells produced by TechnologyTransfer (TT) and Process Qualification (PQ) manufacturing runs showspecific cytotoxic activity, e.g., IFN-γ release, against the targetpositive cells. No IFN-γ release was detected against the negativecontrol cells.

FIG. 42 shows EC₅₀ determination based on IFN-γ response against targetcells pulsed with decreasing concentrations of the cognate peptide. Theresults show T cells produced by Process Qualification (PQ1)manufacturing run exhibit anti-tumor activity (EC₅₀=0.3149) comparableto that produced by the positive control in the assay (EC₅₀=0.7037).

FIG. 43 shows a representative figure of cytotoxic potential of T CellProduct #3 in the Incucyte® killing assay. Data is presented as foldtumor growth in the presence of T Cell Product #3 over 72h co-culturingperiod with a target negative cell line pulsed with decreasingconcentration of the relevant peptide. The results show a peptide dosedependent killing of target cells by T cells produced by ProcessQualification (PQ1) manufacturing run.

In sum, shorter ex-vivo expansion and overall “turnaround time” can havea substantial impact not only on the quality of the cell product butalso clinical applicability of cellular immunotherapies. The processdevelopment efforts to shorten the expansion phase during GMPmanufacturing of TCR engineered T cells were completed with thedevelopment of a robust, 5-6 day long, semi-closed T cell manufacturingprocess for T Cell Product #3. The Technology Transfer (TT) and ProcessQualification (PQ) runs for T Cell Product #3 manufacturing in GMPenvironment cleanroom confirmed the reproducibility and feasibility ofthe manufacturing process with shortened expansion phase. All therelease, phenotype, and functionality testing of the TCR engineered Tcells were confirmed for the GMP manufactured T cell products.

Example 7

Manufacturing and Functionality of T Cell Products Generated from CancerPatients

As noted above, T Cell Product #3 generated from healthy donors showT-cell memory phenotype and cytotoxic potentials. As shown below,similar characteristics were observed in T Cell Product #3 generatedfrom cancer patients, when compared with that of T Cell Product #3generated from healthy donors.

Patient and Donor Characteristics

Disease Patient Status/Chemo (PT)/Donor Primary Clinical TreatmentTreatment Status: (D) diagnosis Age Gender Race Stage Status TreatmentNotes PT1 Ovarian 78 Female W IV Stable/Active Cisplatin/Gemzar CancerTreatment PT2 Ovarian 69 Female W III-C Stable/Active Doxil CancerTreatment PT3 Ovarian 73 Female W III-B Stable/Active Carboplatin/GemzarCancer Treatment PT4 Endometrial 72 Female AI III-A Unknown/Taxol/Carboplatin Cancer Pre-treatment D1 Normal 69 Male W N/A UnknownN/A D2 Normal 70 Male W N/A Unknown N/A D3 Normal 62 Male W N/A UnknownN/A D4 Normal 52 Female H N/A Unknown N/A W = White; AI = AmericanIndian; H = Hispanic

T Cell Product #3 were manufactured in small scale using PBMC obtainedfrom cancer patients and healthy donors. Briefly, on Day 0,cryopreserved PBMC isolated from leukapheresis products of 4 cancerpatients and 4 healthy donors were thawed and rested in the presence ofIL-7 for about 4-6 hours, followed by activation in NTC 24-well platesand incubation for about 16-24 hours. On Day 1, cells transduced withviral vector expressing recombinant TCR, e.g., R11KEA TCR, at 5 μl/10⁶cells. Non-transduced (NT)) cells were included as controls. Transducedand non-transduced cells were seeded at a minimum of 1.0×10⁶ cells/ml,e.g., 2.0×10⁶ cells/ml. On Day 2, Transduced and non-transduced cellswere expanded in TexMACS complete medium with IL-7 and IL-15. On Day 6,i.e., expansion for 4 days, expanded cells were harvested followed byflow cytometry analysis and functional assays to determine, e.g.,recovery, viability, phenotypes, integrated DNA copy numbers, andfunctionality.

FIG. 44 shows comparable recoveries of T cells obtained from cancerpatients (Pt) and healthy donors (HD) at post-thawing, post-resting, andpost-activation.

FIG. 45 shows comparable total viable cells and % viability of T CellProduct #3 on Day 6, i.e., expansion for 4 days, in transduced andnon-transduced cells within each individual, except PT1 and PT4, inwhich all cells were transduced.

FIG. 46 shows comparable fold-expansion of T Cell Product #3 on Day 6,i.e., expansion for 4 days, in transduced and non-transduced cellswithin each individual, except PT1 and PT4, in which all cells weretransduced.

Phenotype Analysis

FIG. 47 shows preferential expansion of CD3+CD8+ cells (as indicated byarrows), as compared with that of CD3+CD4+ cells, in PBMCs obtained fromcancer patients (PT1-PT4) and healthy donors (D1-D4).

FIG. 48 shows comparable overall averages of the CD3+CD8+ cells and theCD3+CD4+ cells in T Cell Product #3 and non-transduced cells (NT)obtained from patients (PT1-PT4) and healthy donors (D1-D4).

FIG. 49 shows an example of flow cytometry analysis of T Cell Product#3. The results indicate 43.8% of T Cell Product #3 contain CD3+CD8+cells, in which 64.7% of the cells expressing R11KEA TCR, as indicatedby peptide/MHC dextramer (Dex) staining, and 35.3% of the cells that donot express R11KEA TCR.

FIG. 50 shows comparable R11KEA TCR expression in CD8+ T Cell Product #3generated from cancer patients (PT1-PT4) and healthy donors (D1-D4).

FIG. 51 also shows comparable average R11KEA TCR expression in CD8+ TCell Product #3 generated from cancer patients (PT1-PT4) (e.g., 64.3%)and healthy donors (D1-D4) (e.g., 68.2%).

FIG. 52 shows gating scheme to determine T cell memory (T_(memory))phenotype of T Cell Product #3. For example, by gating for CD45RA andCCR7, naïve “young” T cells (CD45RA+CCR7+), terminally differentiated“old” T cells (TemRA) (CD45RA+CCR7−), effector memory T cells (Tem)(CD45RA−CCR7−), and central memory T cell (Tcm) (CD45RA−CCR7+) can beidentified.

FIG. 53 shows notable average increases in both desirable naive and Tcmcompartments of T Cell Product #3 generated from both patients (PT1-PT4)and healthy donors (D1-D4). These results suggest that transduced cellsmay possess greater ability to persist after infusion and produce longerlasting responses in vivo.

Functional Assays

To determine the functionality of T Cell Product #3, cells may bestimulated with relevant peptide (e.g., 1 μg/ml) that specifically bindsR11KEA TCR or irrelevant peptide (e.g., 1 μg/ml), which does not bindR11KEA TCR, as a control. Stimulation with PMA and ionomycin, whichactivate all lymphocytes, serves as positive control; andnon-stimulation serves as negative control. After 2 hours ofstimulation, protein transport inhibitors were added. At 6 hours afterstimulation, expression of cytokines and signalling molecules, e.g.,CD107a, IFN-γ, TNF-α, IL-2, and macrophage inflammatory protein-1-beta(MIP-1β), in CD3+CD8+ cells were evaluated by intracellular staining(ICS).

FIG. 54 shows an example of T Cell Product #3, after stimulation withthe relevant peptide (d), the expression levels of CD107a, IFN-γ, TNF-α,IL-2, and MIP-1β in T Cell Product #3 increase as compared with that ofstimulation with the irrelevant peptide (c). Stimulation with PMA andionomycin, which activate all lymphocytes, serves as positive control(b); and non-stimulation serves as negative control (a).

FIG. 55 shows polyfunctionality of T Cell Product #3. The numbers 0, 1,2, 3, 4, and 5 denote, respectively, the portion of T Cell Product #3express none, any one, any two, any three, any four, and all five ofCD107a, IFN-γ, TNF-α, IL-2, and MIP-1β. For example, after stimulationwith relevant peptide, more than 50% of the T Cell Product #3 obtainedfrom healthy donor (D3) transduced with R11KEA TCR (R11) express atleast 2 cytokines from CD107a, IFN-γ, TNF-α, IL-2, and MIP-1β, ascompared with that of stimulation with irrelevant peptide, i.e., 0% ofcells express at least 2 cytokines. In contrast, there is no significantdifference in cytokine expression in non-transduced (NT) cells betweenstimulation with relevant peptide and irrelevant peptide. These resultsshow T Cell Product #3 generated from healthy donors and transduced withR11KEA TCR is polyfunctional. The positive controls, i.e., T cellsstimulated with PMA/ionomycin, exhibit polyfunctionality with or withoutTCR transduction. The negative controls, i.e., T cells withoutstimulation, exhibit poor functionality with or without TCRtransduction.

FIG. 56 shows, after stimulation with relevant peptide,polyfunctionality of the R11KEA TCR+ (CD8+Vb8+) T Cell Product #3generated from healthy donors, e.g., D3 and D2, and from cancerpatients, e.g., PT1, PT2, and PT3. T cells generated from D1, D4, andPT4 may not appear polyfunctional as determined by these functionalassays. As shown below, T cells generated from D1, D4, and PT4, however,still have cytotoxic activity against target cells.

FIG. 57 shows IFN-γ release from T Cell Product #3 generated from cancerpatients, e.g., PT1-PT4, when these cells were in contact with a hightarget cell line, which has about 1,000 copies/cell of the relevantpeptide presented on the cell surface, in E:T ratio-dependent manner,e.g., 10:1>3.3:1>1:1. Note that T cells generated from PT4, which maynot appear polyfunctional in FIG. 56, also show IFN-γ release in E:Tratio-dependent manner.

FIG. 58 shows IFN-γ release from T Cell Product #3 generated fromhealthy donors, e.g., D1-D4, when these cells were in contact with ahigh target cell line, which has about 1,000 copies/cell of the relevantpeptide presented on the cell surface in E:T ratio-dependent manner,e.g., 10:1>3.3:1>1:1. Note that T cells generated from D1 and D4, whichmay not appear polyfunctional in FIG. 56, also show IFN-γ release in E:Tratio-dependent manner.

FIG. 59 shows average IFN-γ release from T Cell Product #3 generatedfrom healthy donors (D1-D4), when these cells were in contact with cellswith different levels of relevant peptide presented on the cell surface,e.g., high-target cell line that has about 1,000 copies/cell of therelevant peptide presented on the cell surface, low-target cell linethat has about 50 copies/cell of the relevant peptide presented on thecell surface, and none-target cell line that does not have the relevantpeptide presented on the cell surface, in peptide presentationlevel-dependent manner, i.e., high-target>low-target>none-target.

FIG. 60 shows, similarly, average IFN-γ release from T Cell Product #3generated from cancer patients (PT1-PT4), when these cells were incontact with high-target cell line, low-target cell line, andnone-target cell line, in peptide presentation level-dependent manner,e.g., high-target>low-target>none-target.

FIG. 61 shows lack of killing activity of T Cell Product #3 generatedfrom healthy donor, e.g., D3, in contact with target-negative cell line,in which the relevant peptide is not presented on the cell surface.Briefly, T cells generated from D3 transduced with R11KEA TCR (R11) orwithout transduction (NT) were co-cultured with target-negative cellline at E:T ratios of 10:1, 3.3:1, and 1:1. Cell killing activity wasmeasured by using IncuCyte Killing Assay. These results show nosignificant difference in cell killing against target-negative cell linebetween T cells with (R11) and without (NT) TCR transduction.

In contrast, FIG. 62 shows TCR-specific killing activity of T CellProduct #3 generated from healthy donor, e.g., D3, in contact withtarget-positive cell line, in which the relevant peptide is presented onthe cell surface. That is, R11KEA TCR-expressing T cells kill thetarget-positive cells in E:T ratio-dependent manner, e.g.,10:1>3.3:1>1:1. In contrast, there is no significant difference in cellkilling between T cells without transduction (NT) at different E:Tratios.

FIGS. 63A-63C show TCR-specific killing activity of T Cell Product #3transduced with R11KEA TCR (R11) generated from healthy donors, e.g., D3and D4, and from cancer patients, e.g., PT1 and PT2, in contact withtarget-positive cell line, in which the relevant peptide is presented onthe cell surface. R11KEA TCR (R11)-expressing T cells generated from D3,D4, PT1, and PT2 kill the target-positive cells in E:T ratio-dependentmanner, e.g., 10:1 (FIG. 63A)>3.3:1 (FIG. 63B)>1:1 (FIG. 63C). Incontrast, there is no significant difference in cell killing between Tcells without R11KEA TCR transduction (NT) at different E:T ratios.

In sum, these results show T Cell Product #3 process may generate T cellproducts expressing TCR transgene with target specificity. This processworks as well with starting material obtained from cancer patients asfrom healthy donors. T Cell Product #3 process takes shorter time thanthat for preparing T Cell Products #1 and #2 and yet generates productswith large numbers of naïve and Tcm cells. T Cell Product #3 may bepolyfunctional and secrete IFN-γ in response to target-positive tumorcell lines. T Cell Product #3 may also exhibit good effector function incell line killing assays.

Advantages of the present disclosure may include autologous T cellmanufacturing processes that may shorten resting time to, e.g., 4-6hours, activation time to, e.g., 16-20 hours, transduction time to,e.g., 24 hours, and expansion phase to, e.g., 5-7 days, for clinicalmanufacturing of engineered TCR T cell products. Critical parametersinfluencing each step may be systematically evaluated and may beoptimized to yield over 10 billion young, tumor-reactive T cells with astrong ability to recognize and efficiently kill target expressing tumorcells. In addition to improving the quality of T cell products, theseoptimizations may also result in reducing the cost of manufacturing by30%. Further, autologous T cell manufacturing processes of the presentdisclosure may be scaled up using flask bound and/or bag bound anti-CD3and anti-CD28 antibodies for activating T cells to yield comparablelevels of activation, transducibility, and expansion and these scale-upprocesses may be faster than processes using plate bound antibodies.

All references cited in this specification are herein incorporated byreference as though each reference was specifically and individuallyindicated to be incorporated by reference. The citation of any referenceis for its disclosure prior to the filing date and should not beconstrued as an admission that the present disclosure is not entitled toantedate such reference by virtue of prior invention.

It will be understood that each of the elements described above, or twoor more together may also find a useful application in other types ofmethods differing from the type described above. Without furtheranalysis, the foregoing will so fully reveal the gist of the presentdisclosure that others can, by applying current knowledge, readily adaptit for various applications without omitting features that, from thestandpoint of prior art, fairly constitute essential characteristics ofthe generic or specific aspects of this disclosure set forth in theappended claims. The foregoing embodiments are presented by way ofexample only; the scope of the present disclosure is to be limited onlyby the following claims.

What is claimed is:
 1. A method of transducing a T cell populationcomprising thawing frozen peripheral blood mononuclear cells (PBMC),resting the thawed PBMC, activating the T cells in the rested PBMC withan anti-CD3 antibody and anti-CD28 antibody, transducing the activated Tcells with a viral vector, expanding the transduced T cells, andobtaining the expanded T cells, wherein the expanded T cells are capableof specifically binding a peptide consisting of the amino acid sequenceof SLLMWITQC (SEQ ID NO: 131) or GVYDGREHTV (SEQ ID NO: 89).
 2. Themethod of claim 1, wherein the activation further comprises incubationwith IL-2.
 3. The method of claim 1, wherein the IL-2 concentration isbetween about 50 IU/mL and 150 IU/mL.
 4. The method of claim 1, whereinthe viral vector is a lentivirus vector.
 5. The method of claim 1,wherein the T cells are expanded for 1-15 days.
 6. The method of claim1, wherein the T cells are expanded in the presence of IL-2, IL-7,IL-12, IL-15, or a combination thereof.
 7. The method of claim 1,wherein the T cells are CD4+.
 8. The method of claim 1, wherein the Tcells are CD8+.
 9. A method of treating a patient having a cancercomprising administering a composition comprising the T cell of claim 1.10. The method of claim 9, wherein the cancer is melanoma, ovariancancer, esophageal cancer, non-small cell lung cancer (NSCLC), or acombination thereof.
 11. The method of claim 9, the T cells areautologous.
 12. The method of claim 9, wherein the patient is HLA-A*02.13. The method of claim 9, wherein the dosage of the T-cells is about1×10⁶ to about 1×10⁹ transduced T cells/m² (or kg) of the patient. 14.The method of claim 9, wherein the T-cells are administered viacontinuous infusion.
 15. The method of claim 1, wherein the expanded Tcells are capable of specifically binding a peptide consisting of theamino acid sequence of SLLMWITQC (SEQ ID NO: 131).
 16. The method ofclaim 15, wherein the viral vector comprises a nucleic acid encoding a Tcell receptor (TCR) that binds a peptide consisting of the amino acidsequence of SLLMWITQC (SEQ ID NO: 131).
 17. The method of claim 1,wherein the expanded T cells are capable of specifically binding apeptide consisting of the amino acid sequence of GVYDGREHTV (SEQ ID NO:89).
 18. The method of claim 17, wherein the viral vector comprises anucleic acid encoding a TCR that binds a peptide consisting of the aminoacid sequence of GVYDGREHTV (SEQ ID NO: 89).
 19. The method of claim 6,wherein the T cells are expanded in the presence of IL-7.
 20. The methodof claim 6, wherein the T cells are expanded in the presence of IL-15.